NL1031818C2 - New antibody or antigen-binding portion that competes for binding to P-cadherin or binds to the same epitope of P-cadherin, useful for treating abnormal cell growth, including cancer, e.g. prostate cancer or bladder cancer - Google Patents

Abstract

An antibody or antigen-binding portion that increases spheroid disruption in a P-cadherin-dependent spheroid disruption assay, competes for binding to P-cadherin, or binds to the same epitope of P-cadherin, is new. A new antibody or antigen-binding portion has at least one property selected from: (a) a K d(E)/K d(P) that is greater than or equal to 1.5; (b) binds to P-cadherin with a K d of 50 nM or less as measured by surface plasmon resonance; (c) a k off for P-cadherin of less than or equal to 0.01 s-1> as measured by surface plasmon resonance; (d) an IC 50 of 50 nM or less as measured by a P-cadherin dependent cell adhesion assay; (e) an IC 50 of 50 nM or less as measured by a P-cadherin dependent cell aggregation assay; (f) increases spheroid disruption in a P-cadherin-dependent spheroid disruption assay by a factor of 2 or more as compared to a control sample with no IgG; (g) competes for binding to P-cadherin with an antibody selected from 194-e06; 194-a02; 194-b09; 195-e11; 194-g09; 196-h02; 194-e01; 196-d10; 196-g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4; or g-129-1c4; (h) cross-competes for binding to P-cadherin with an antibody selected from 194-e06; 194-a02; 194-b09; 195-e11; 194-g09; 196-h02; 194-e01; 196-d10; 196-g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06129-1c4; or g-129lc4; (i) binds to the same epitope of P-cadherin as an antibody selected from 194-e06; 194-a02; 194-b09; 195-e11; 194-g09; 196-h02; 194-e01; 196-d10; 196-g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4; or g-129-1c4; (j) binds to P-cadherin with substantially the same K d as an antibody selected from 194-e06; 194-a02; 194-b09; 195-e11; 194-909; 196-h02; 194-e01; 196-d10; 196-g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4; or g-129-1c4; or (k) binds to P-cadherin with substantially the same koff as an antibody selected from 194-e06; 194-a02; 194-b09; 195-e11; 194-g09; 196-h02; 194-e01; 196-d10; 196-g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4; and g-129-1c4. ACTIVITY : Cytostatic. No biological data given. MECHANISM OF ACTION : Gene therapy; P-cadherin antagonist.

Description

P-CADHERXNE ANTIBODIES

This application invokes the priority of US Provisional Application 60 / 675,311, filed April 26, 2005, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen-binding portions thereof that bind to β-cadherin. The invention also relates to nucleic acid molecules encoding such antibodies and antigen-binding portions, methods of making β-cadherin antibodies and antigen-binding portions, compositions comprising these antibodies and antigen-binding portions, and methods of using the antibodies, antigen-binding portions and preparations.

Background of the Invention Cadherins are a superfamily of transmembrane glycoproteins that regulate cell-cell adhesion during development and tissue homeostasis (Gumbiner, J. Cell. Biol., 148: 399-404 (2000); Yagi et al., Genes Dev. , 14: 1169-1180 (2000)). The intracellular domains of cadherins interact with cytoplasmic proteins such as catenins and p120, which form the basis of cadherin attachment to the actin cytoskeleton. Cadherins have five extracellular Ca 2+ binding domains and a small cytoplasmic domain that is highly conserved among the classical cadherins. Members of the classical cadherin family include P-cadherin, E-cadherin and N-cadherin. Cellular adhesion molecules such as cadherins are thought to play an important role in the cellular compounds of cancer and metastatic cells (Furukawa et al., Microscopy Res. Technique 38 (4): 343-352 (1997)). Β-cadherin expression in normal adult tissues is low and is mainly limited to myo-epithelial cells and the basal layers of layered epithelium (Shimoyama et al., Cancer Res. 49: 2128-33 (1989)). P-cadherin is up-regulated in bowel diseases with inflammatory process such as Crohn's disease and colitis (Hardy et al., Gut 50: 513-519 (2002)). A large amount of evidence now also reveals that abnormal β-cadherin expression is associated with cell proliferation and with tumors of the colon, breast, lung, thyroid and cervix (Gamallo, Modern Pathology, 14: 650-654, ( 2001) and Stefansson et al., J. Clin. Oncol. 22 (7): 1242-15 1252 (2004)). Human β-cadherin has been reported to be the antigen recognized by the NCC-CAD-299 monoclonal antibody raised against epidermoid carcinoma of the vulva (Shimoyama et al., Cancer Res., 49: 2128-2133 ( 1989)). It is expected that modulation of β-cadherin-mediated adhesion and intracellular signaling leads to reduced proliferation and survival of tumor cells in vivo. Accordingly, in view of the central role that P-cadherin appears to play in cell proliferation and solid tumor progression, it is desirable to generate antibodies to P-cadherin that can provide a therapeutic benefit for patients with a variety of cancers .

Summary of the Invention In one aspect of the present invention, a P-30 cadherin antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof has at least one of various functional properties, as described below in A) to K).

A) For example, in one embodiment, the antibodies or antigen-binding portions thereof have a greater binding affinity for β-cadherin (Kq (P)) than for β-cadherin (K ^ E)). In one embodiment, the antibodies or antigen-binding portions thereof of the present invention have a KD (E) / KD (P) that is greater than or equal to 1.5. In a further embodiment, the antibodies or antigen-binding portions thereof of the present invention have a Κρ (Ε) / Κβ (Ρ) that is greater than, or equal to 2, greater than, or equal to 3, greater than, or equal to 5 , greater than, or equal to 10, greater than, or equal to 20, greater than, or equal to 50, greater than 10, or equal to 100, greater than, or equal to 200, greater than, or equal to 500 or greater than, or equal to 1000. There is typically no upper limit with respect to the value of KD (E) / KD (P) because the KdCE) value can be very small, such as 0. For practical purposes, an upper limit of K ^ Ei / K ^ P) can be however, 1 x 106. Such KD values for both P-cadherin and for E-cadherin can be measured by any technique known to those skilled in the art, such as by ELISA, RIA, flow cytometry or surface plasma monononance, such as BIACORE ™.

B) In another embodiment, the antibody or portion thereof binds to β-cadherin with a Kj, of 1000 nM or less, as measured by surface plasma resonance. In a further embodiment, the antibody or portion binds to P-cadherin with a KD of less than 50 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 500 pM or less than 100 pM, as measured by surface plasmon resonance. There is typically no lower limit with respect to the value of Ko. For practical purposes, however, it can be assumed that the lower limit is about 1 pM.

C) In another embodiment, the antibody or portion thereof has an β-cadherin β-cadherin reduction rate of less than or equal to 0.01 µl as measured by surface plasma monononance. For example, in certain embodiments, the antibody or moiety 4 has a Kaβ for β-cadherin of less than 0.005 s'1, less than 0.004 s'1, less than 0.003 s'1, less than 0.002 s'1 or less than 0, There is typically no lower limit for the value of Ka £. For practical purposes, however, it can be assumed that the lower limit is about 1 x 10 7 6-1.

D) In another embodiment, the β-cadherin antibody or portion thereof has an IC 50 of 100 nM or less, as measured by a P-10 cadherin-dependent cell adhesion assay. In a further embodiment, that IC 50 is less than 50 nM, less than 40 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 200 pM, less than 100 pM or less than 10 pM, as measured by a P-15 cadherin-dependent cell adhesion assay. There is typically no lower limit for the value of IC 50 as measured by a P-cadherin dependent cell adhesion assay. For practical purposes, however, it can be assumed that the lower limit is about 1 pM.

E) In another embodiment, the P-cadherin antibody or portion thereof has an IC50 of 100 nM or less, as measured by a P-cadherin-dependent cell aggregation assay. In a further embodiment, that IC 50 is less than 50 nM, less than 40 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 200 pM, less than 100 pM or less than 1 pM, as measured by a P-cadherin-dependent cell aggregation assay. There is typically no lower limit for the value of IC50 as measured using a P-cadherin-dependent cell aggregation assay. For practical purposes, however, it can be assumed that the lower limit is about 1 pM.

F) In another embodiment, the β-cadherin antibody or portion thereof increases spheroid disruption in a β-cadherin-dependent spheroid disruption assay by a factor of at least 2 compared to a control sample with no IgG present. In a further embodiment, the β-cadherin antibody or portion thereof increases spheroid disruption in a β-cadherin-dependent spheroid disruption assay by a factor of at least 3, at least 4, at least 6, at least 10 or at least 15, compared to a contro-lemon star without IgG present.

G) In another embodiment, the β-cadherin antibody or portion thereof competes with an antibody selected from the group consisting of 194-e06; 10 194 -a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 19 6-g 0 3; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b 9; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-lc4 and g-129-lc4, for binding to β-cadherin.

H) In another embodiment, the β-cadherin antibody or portion thereof cross-competes with an antibody selected from the group consisting of 194-e06; 194 -a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 19 6-g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-2006; 129-lc4 and g-129-lc4, for binding to β-cadherin.

I) In another embodiment, the β-cadherin antibody or portion thereof binds to the same epitope of β-cadherin as an antibody selected from the group consisting of 194-e06; 194-a02; 194-b09; 195-25 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 196-e06; 19 5-a 0 9; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4 and g-129-1c4.

J) In another embodiment, the P-30 cadherin antibody or portion thereof binds to P-cadherin with substantially the same KD as an antibody selected from the group consisting of 194-e06; 194-a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4 and g-129-1C4.

K) In another embodiment, the β-cadherin antibody or portion thereof binds to β-cadherin with substantially the same Kaβ as an antibody selected from the group consisting of 194-e06; 194-a02; 5 194-bO 9; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 19 6 -e 0 6; 195-a09; 198-aO9; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4 and g-129-1c4.

A further aspect of the present invention is an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), comprising a VH domain that is at least 90% identical in amino acid sequence to one of SEQ ID NOs: 1 to 13 and 320 to 15 325. In one embodiment, that amino acid sequence VH domain is at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% identical to one of SEQ ID NOs: 1 to 12 and 320 to 325.

In a further embodiment, the antibody or portion thereof has at least one of the functional properties previously described in A) to K), comprising a VH domain consisting of one of SEQ ID NOs: 1 to 13 and 320 to 325, or different from one of SEQ ID NOs: 1 to 13 and 320 to 325 by having at least one conservative amino acid substitution. For example, the VH domain may differ by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions from one of SEQ ID NOs: 1 to 13 and 320 to 325. In a further embodiment, which of these conservative amino acid substitutions also occur in the CDR1, CDR2 and / or CDR3 regions.

A further aspect of the present invention is an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), comprising a VL domain that has at least 90% amino acid sequence is identical to one of SEQ ID NOs: 14 to 23 and 326 to 7 331. In one embodiment, that VL domain in amino acid sequence is at least 91%, at least 93%, at least 95%, at least 97%, at least at least 99% or 100% identical to one of SEQ ID NOs: 14 to 23 and 326 to 331.

In a further embodiment, the antibody or portion thereof has at least one of the functional properties previously described in A) to K), and includes a V ^ domain consisting of one of SEQ ID NOs: 14 to 23 and 326 to 331, or differs from one of SEQ ID NOs: 14 to 23 and 326 to 331 by having at least one conservative amino acid substitution. For example, the VL domain can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions from one of SEQ ID NOS: 14 to 23 and 326 to 331. In a further embodiment, any of these conservative amino acid substitutions occur in the CDR1, CDR2 and / or CDR3 regions.

Another aspect of the present invention is an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), wherein the VL and VH domains each have at least amino acid sequence in terms of amino acid sequence 90% are identical to the VL and VH domains of one of antibodies 194-e06, respectively; 194-a02; 194-b09; 195-25 ell; 19 4 -g 0 9; 196-h02; 194-eOl; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1C4 and g-129-1c4. For example, the VL and VH domains are each at least 91%, 93%, 95%, 97%, 99% or 100% identical to the VL and VH domains of one of antibodies 194-e06, respectively, with respect to amino acid sequences ; 194-a02; 194-bO 9; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 196-06; 19 5 -a 0 9; 198-a09; 200-hO 6; g-194-b 9; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1C4 and g-12 12 9 -c4.

In another aspect of the present invention, an antibody or antigen-binding portion thereof that is b is selected from the group consisting of: a) an antibody or portion thereof comprising a VH domain, as set forth in SEQ ID NO: 1, and a VL domain as set forth in SEQ ID NO: 14; b) an antibody or portion thereof comprising a V H domain as set forth in SEQ ID NO: 2 and a V L domain as set out in SEQ ID NO: 14; c) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 2 and a VL domain as set forth in SEQ ID NO: 15;

d) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 3 and a VL domain as set forth in SEQ ID NO: 16; e) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 4 and a VL domain as set forth in SEQ

ID NO: 17; f) an antibody or portion thereof

that includes a VH domain as set forth in SEQ ID NO: 4 and a VL domain as set forth in SEQ ID NO: 23; g) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 5 and a VL domain as set forth in SEQ ID NO: 18; h) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 6 and a VL domain as set forth in SEQ

ID NO: 23; i) an antibody or portion thereof comprising a V H domain, as set forth in SEQ ID NO: 7, and a V L domain, as set out in SEQ ID NO: 23;

j) an antibody or portion thereof comprising a V H domain, as set forth in SEQ ID NO: 8, and a V L domain, as set out in SEQ ID NO: 23; k) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 9 and a VL domain as set out in SEQ

ID NO: 23; 1) an antibody or portion thereof comprising a V H domain as set forth in SEQ ID NO: 10 and a V L domain as set out in SEQ ID NO: 19; m) an antibody or portion thereof comprising a V H domain, as set forth in SEQ ID NO: 11, and a V L domain, as set out in SEQ ID NO: 20; n) an antibody or portion thereof comprising a V H domain as set forth in SEQ ID NO: 12 and a V L domain as set out in SEQ ID NO: 21; o) an antibody or portion thereof comprising a V H domain as set forth in SEQ ID NO: 13 and a V L domain as set out in SEQ ID NO: 5 22; p) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 320, and a VL domain as set forth in SEQ ID NO: 326; q) an antibody or antigen-binding portion thereof comprising a VH domain, as set forth in SEQ ID NO: 321, and a VL-10 domain, as set forth in SEQ ID NO: 327; r) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 322 and a VL domain as set forth in SEQ ID NO: 328; s) an antibody or portion thereof comprising a V H domain, as set forth in SEQ ID NO: 323, and a V L domain, as set out in SEQ ID NO: 329; t) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 324 and a VL domain as set out in SEQ ID NO: 330; and u) an antibody or portion thereof comprising a VH domain as set forth in SEQ ID NO: 325 and a VL domain as set forth in SEQ ID NO: 331.

In a further embodiment, for any of the antibodies or portions thereof as described above in groups a) to u), the V H and / or V L domains may differ from the specific SEQ ID NOs recited therein by at least at least one conservative amino acid substitution. For example, the VH and / or VL domains can differ from the recited SEQ ID NO with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions. In a further embodiment, any of these conservative amino acid substitutions may occur in the CDR1, CDR2 and / or CDR3 regions.

In another embodiment, the present invention provides an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), comprising a VH domain independently selected from one of SEQ ID NOs: 1 to 13 and 320 to 325, or a sequence that differs from one of SEQ ID NOs: 1 to 13 and 320 to 325 with at least one conservative amino acid substitution, and further comprises a VL domain that is independent of 5 is selected from one of SEQ ID NOs: 14 to 23 and 326 to 331, or a sequence different from one of SEQ ID NOs: 14 to 23 and 326 to 331 with at least one conservative amino acid substitution. For example, the VH and VL domains can each independently differ from one of SEQ ID NOs: 1 to 13, 320 to 325, 14 to 23 and 326 to 331 with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

In a further embodiment, the present invention provides an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), wherein said antibody or portion comprises a VH-CDR3 selected from one of SEQ ID NOs: 26 to 37 and 91 to 256, or a sequence that differs from one of SEQ ID NOs: 26 to 37 and 91 to 256 with at least one conservative amino acid substitution. For example, the VH-CDR3 can differ from one of SEQ ID NOs: 26 to 37 and 91 to 256 with 1, 2, 3 or 4 conservative amino acid substitutions.

In a further embodiment, the present invention provides an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), wherein said antibody or antigen-binding portion comprises a VL-CDR3, selected from one of SEQ ID NOs: 40 to 47 and 257 to 319, or a sequence different from one of SEQ ID NOs: 40 to 47 and 257 to 319 with at least one conservative amino acid substitution. The VL-CDR3 can, for example, differ from one of SEQ ID NOs: 40 to 47 and 2 57 to 319 with 1, 2, 3, 4 or 5 conservative amino acid substitutions.

In a further embodiment, the present invention provides an antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion: a V1 CDR1 as set forth in SEQ ID NO: 24, or a sequence different from SEQ ID NO: 24 with at least one conservative amino acid substitution; a VH-CDR2 as set forth in SEQ ID NO: 25 or a sequence different from SEQ ID NO: 25 with at least one conservative amino acid substitution; and a VH-CDR3 independently selected from one of SEQ ID NOs: 26 to 37 and 91 to 256, or a sequence different from one of SEQ ID NOs: 6 to 37 and 91 to 256 with at least one conservative amino acid substitution. For example, the above-mentioned VH-CDR1, -CDR2 and -CDR3 sequences may each independently differ from the respective recited SEQ ID NOs with 1, 2, 3, 4 or 5 conservative amino acid substitutions.

In another embodiment, the present invention provides an antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion: a VL-CDR1 as set forth in SEQ ID NO: 38, or a sequence different from SEQ ID NO: 38 with at least one conservative amino acid substitution; a VL-CDR2 as set forth in SEQ ID NO: 39, or a sequence different from SEQ ID NO: 39 with at least one conservative amino acid substitution; and a VL-CDR3 independently selected from one of SEQ ID NOs: 40 to 47 and 257

to 319, or a sequence that differs from one of SEQ ID

NOs: 40 to 47 and 257 to 319 with at least one conservative amino acid substitution. For example, the above-mentioned VL-CDR1, -CDR2 and -CDR3 sequences may differ independently from the respective recited SEQ ID NOs with 1, 2, 3, 4 or 5 conservative amino acid substitutions.

The present invention further provides an antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion: a VH-CDR1 as set forth in SEQ ID NO: 24; a VH-CDR2 as set forth in SEQ ID NO: 25; a VH-CDR3 selected from i2 one of SEQ ID NOs: 26 to 37 and 91 to 256; a VL-CDR1 as set forth in SEQ ID NO: 38; a Vb CDR2 as set forth in SEQ ID NO: 39; and a Vb-CDR3 selected from one of SEQ ID NOs: 40 to 47 and 257 to 319, including 5. In a further embodiment, the stated VH and V1 CDR1, CDR2 and CDR3 sequences may also each independently differ from the specific SEQ ID NOs recited above with at least one conservative amino acid substitution. For example, the CDR1, CDR2 and CDR3 sequences may each independently differ from the respective specific SEQ ID NOs recited above with 1, 2, 3, 4 or 5 conservative amino acid substitutions,

The present invention further provides an antibody or antigen-binding portion thereof wherein said antibody or antigen-binding portion comprises the VH and Vb-CDR1, the VH and Vb-CDR2 and the VH and Vb-CDR3 as found in any of antibodies 194-e06; 194-a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 19 6 -e 0 6; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-20 g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1C4 and g-129-lc4.

The present invention further provides an antibody or antigen-binding portion thereof, comprising a VH domain selected from one of SEQ ID NOs: 1 to 13 and 320 to 325, or different from one of SEQ ID

NOs: 1 to 13 and 320 to 325 with 1 to 10 conservative amino acid substitutions.

The present invention further provides an antibody or antigen-binding portion thereof, comprising a Vb domain selected from one of SEQ ID NOs: 14 to 23 and 326 to 331, or different from one of SEQ ID

NOs: 14 to 23 and 326 to 331 with 1 to 10 conservative amino acid substitutions.

The present invention further provides an antibody or antigen-binding portion thereof, comprising a VH domain independently selected from one of SEQ ID NOs: 1 to 13 and 320 to 325, or a sequence that is 13 different from one of SEQ ID NO1 s: 1 to 13 and 320 to 325 with 1 to 10 conservative amino acid substitutions, and further comprising a V L domain independently selected from one of SEQ ID NOs: 14 to 23 and 326 to 331, or a sequence that differs from one of SEQ ID NOs: 14 to 23 and 326 to 331 with 1 to 10 conservative amino acid substitutions.

The present invention further provides an antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion: a VH-CDR1 as set forth in SEQ ID NO: 24, or a sequence different from SEQ ID NO: 24 by 1 to 4 conservative amino acid substitutions; a VH-CDR2 as set forth in SEQ ID NO: 25, or a sequence that differs from SEQ ID NO: 25 with 1 to 4 conservative amino acid substitutions; and a

Vh-CDR3 selected from one of SEQ ID NOs: 26 to 37 and 91 to 256, or a sequence different from one of SEQ ID NOs: 26 to 37 and 91 to 256 with 1 to 4 conservative amino acid substitutions.

The present invention further provides an antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion: a VL-CDR1 as set forth in SEQ ID NO: 38, or a sequence different from SEQ ID NO: 38 with 1 to 4 conservative ami -

no-acid substitutions; a VL-CDR2 as set forth in SEQ

ID NO: 39, or a sequence different from SEQ ID NO: 39 with 1 to 4 conservative amino acid substitutions; and a

VI-CDR3 selected from one of SEQ ID NOs: 40 to 47 and 257 to 319, or a sequence different from one of 30 SEQ ID NOs: 40 to 47 and 257 to 319 with 1 to 4 conservative amino acid substitutions, includes .

The present invention further provides an antibody or antigen-binding portion thereof with at least one of the functional properties previously described in A) to K), said antibody or antigen-binding portion: a Vh-FR1 as set forth in SEQ ID NO: 48; a V 1 -FR 2 as set forth in SEQ ID NO: 14 49; a VH-FR3 selected from one of SEQ IE NO1s: 50 to 55; a VH-FR4 selected from one of SEQ ID NOs: 56 and 57; a Vtj-FR1 selected from one of SEQ ID NOs: 58 and 59; a VL-FR2 selected from one of SEQ ID NOs: 60 to 62; a VL-FR3 selected from one of SEQ ID NOs: 63 to 66; and a VL-FR4 as set forth in SEQ ID NO: 67. In a further embodiment, the stated VH and VL-FR1, -FR2, -FR3 and -FR4 sequences may also each independently differ from the specific SEQ ID NOs recited above with at least one conservative amino acid substitution. For example, the FR1, FR2, FR3 and FR4 sequences may each independently differ from the respective specific SEQ ID NOs recited above by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. conservative amino acid substitutions. In a still further embodiment, each of the FR1, FR2, FR3 and FR4 sequences may each be mutated independently, thereby matching the respective germline frame sequence.

In a further embodiment of the present invention, one of the antibodies described herein is wherein the antibody is, or is derived from, an IgG, an IgM, an IgE, an IgA, or an IgD molecule. The antibody can be, for example, an IgG2 or IgG2. For example, in one embodiment, IgG is an IgG1f wherein the constant region of the heavy chain comprises SEQ ID NO: 344 and wherein the constant region of the light chain comprises SEQ ID NO: 345, provided that the C-terminal ly -sine residue of SEQ ID NO: 344 is possibly split.

Another embodiment provides one of the antibodies or antigen-binding portions described above that a Fab fragment, an F (ab ') 2 fragment, an Fv fragment, a single-chain Fv fragment, a VH fragment single chain, a single chain VL fragment, a humanized antibody, a chimeric antibody, or a bispecific antibody.

In a further embodiment, a derivatized antibody or antigen-binding portion is one of the antibodies or portions thereof, as described herein, and at least one additional molecular entity. The at least one additional molecular entity may include, for example, another antibody (e.g., a bispecific antibody or diabody), a detection agent, a marker, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that associates the associa-10. antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag). Useful detection agents with which an antibody or antigen-binding portion of the invention may be derivatized include, for example, fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. An antibody may also be labeled with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. In a further embodiment, the antibodies or portions thereof of the present invention may also be labeled with biotin, or with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope drawer -bels). In a still further embodiment of the present invention, one of the antibodies or portions thereof may also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group.

In some embodiments, the β-cadherin antibodies or antigen-binding portions described herein are attached to a solid support.

In some embodiments, the C-terminal heavy-chain lysine is cleaved from one of the β-cadherin antibodies of the invention. In various embodiments of the invention, the heavy and light chains of the β-cadherin antibodies may optionally include an N-terminal signal sequence. The signal sequence of the heavy chain can be, for example, SEQ ID NO: 346, and the signal sequence of the light chain can be SEQ ID NO: 347.

In a further embodiment, the present invention relates to any of the antibodies or antigen-binding portions thereof, as described herein, wherein the antibodies or antigen-binding portions thereof are of human origin.

The present invention also provides a pharmaceutical composition comprising one of the antibodies or antigen-binding portions thereof, as described above, and a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes one of the antibodies or antigen-binding portions thereof, as described herein. In one specific embodiment, an isolated nucleic acid molecule comprising a nucleotide sequence as set forth in one of SEQ ID NOs: 68 to 90 and 332 to 343. The invention further relates to a vector comprising one of the nucleic acid molecules described herein wherein the vector optionally comprises an expression regulatory sequence operably linked to the nucleic acid molecule.

Another embodiment provides a host cell 30 which comprises one of the vectors described herein or which comprises one of the nucleic acid molecules described herein. The present invention also provides an isolated cell line that produces one of the antibodies or antigen-binding portions, as described herein, or that produces the heavy chain or light chain of one of those antibodies or that antigen-binding portions.

In another embodiment, the present invention relates to a method for producing a β-cadherin antibody or antigen-binding portion thereof, comprising culturing one of the host cells or cell lines described herein under suitable conditions and recovering that antibody or antigen-binding portion.

The present invention also relates to a non-human transgenic animal or a transgenic plant comprising one of the nucleic acids described herein, wherein the non-human transgenic animal or the transgenic plant expressing nucleic acid.

The present invention further provides a method for isolating an antibody or antigen-binding portion thereof that binds to β-cadherin, comprising the step of isolating the antibody from the non-human transgenic animal or transgenic plant, as herein described.

The present invention also provides a method for treating abnormal cell growth in a mammal in need thereof, comprising the step of administering to that mammal one of the antibodies or antigen-binding portions thereof, or one of the pharmaceutical preparations as described herein. The present invention further provides a method for treating abnormal cell growth in a mammal in need thereof, with an antibody or antigen-binding portion thereof that binds to β-cadherin, comprising the steps of administering an effective drug to that mammal. A plurality of any of the nucleic acid molecules described herein under suitable conditions that allow expression of those nucleic acid molecules. In another embodiment, the method for treating abnormal cell growth further comprises administering an amount of one or more substances selected from anti-tumor agents, antiangiogenesis agents, signal transduction inhibitors, and anti-proliferative agents, which amounts together are effective at treating that abnormal cell growth. In specific embodiments, that abnormal cell growth is carcinomatous.

The present invention further provides a method for reducing β-cadherin-dependent cellular aggregation, comprising contacting the cells with one of the antibodies or antigen-binding portions thereof described herein, or one of the pharmaceutical compositions described herein .

Another aspect of the present invention is an antibody or antigen-binding portion thereof that comprises an amino acid sequence of a variable region of a heavy chain that uses a human V3 -3 family gene. For example, in one embodiment, the human VH-3 family gene is VH-3-23.

Another aspect of the present invention provides one of the antibodies or antigen-binding portions thereof, as described herein, wherein said antibody or antigen-binding portion is a human antibody. In a further aspect, that antibody or antigen-binding portion is a human recombinant antibody.

The invention also provides a method for producing a β-cadherin antibody or antigen-binding portion thereof, comprising the steps of synthesizing a library of human antibodies in phage, screening the library with β-cadherin or an antigenic portion thereof, isolating phage that binds β-cadherin, and obtaining the antibody from the phage.

Definitions and general techniques

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms will include pluralities and plural terms will include the singular form. Usually, the nomenclature used in connection with, and cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization techniques described herein are those described in generally known and commonly used in the art.

The methods and techniques of the present invention are usually performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout the present description, unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2,000); Ausubel et al., Short Protocols in Molecular Biology; A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Spring, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc.

(2003), the descriptions of which are incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as usually accomplished in the art or as described herein. The nomenclature used in connection with the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known in the art and commonly used. Standard techniques are used for chemical syntheses, chemical analyzes, pharmaceutical preparation, formulation, delivery, and treatment of patients.

The basic structural unit of an antibody is known to include a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The aminoterminal portion of each chain comprises a variable region of about 100 to 120 or more amino acids that is primarily responsible for antigen recognition. The carboxy terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. Inside light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, the heavy chain also comprising a "D" region of about 3 or more amino acids. See Fundamental Immunology in general, Η. 7 (Paul, W., ed., 2e ed., Raven Press, N.Y. (1989)) (here incorporated by reference in its entirety for all purposes). The variable regions of each heavy / light chain pair (VH and Vj form the binding site of the antibody. Thus, for example, an intact IgG antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are the same.

The variable regions of the heavy and light chains exhibit the same general structure of relatively conserved framework regions (FR), connected by three hypervariable regions, also called complementarity determining regions or CDR1s. The term "variable" refers to the fact that, among antibodies, certain portions of the variable domains differ extensively in sequence, and are used in the binding and specificity of each specific antibody for its specific antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies, but is concentrated in the CDRs, which are separated by the more highly conserved FRs. The CDRs from the two chains of each pair are aligned by the FRs, thereby allowing binding to a specific epitope. From N-terminal 5 to C-terminal, both the light and the heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acids are assigned to each domain according to the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Be-10esda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol. , 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989), the disclosures of which are incorporated herein by reference.

It will be understood that the following terms, unless otherwise indicated, have the following meanings:

Unless specifically indicated otherwise, the term "β-cadherin" refers to human β-cadherin, which is an integral membrane protein and a member of the classical cadherin family of transmembrane glycoproteins that regulate cell-cell adhesion. The cloning and sequence of human β-cadherin has been reported, e.g., Shimoyama et al., J. Cell Biol., 109 (4 Pt 1), 1787-1794 (1989), the disclosure of which is incorporated herein by reference. The term P-cadherin is meant to include recombinant human P-25 cadherin and recombinant chimeric forms of P-cadherin, which can be prepared using standard recombinant expression methods, or can be purchased commercially (R&D Systems 861-PC-100 ).

Unless specifically indicated otherwise, the term "E-cadherin" as used herein refers to human E-cadherin, which is an integral membrane protein and a member of the classical cadherin family of transmembrane glycoproteins that regulate cell-cell adhesion. E-cadherin is described, for example, in Takeichi, Science 251: 1451- 1455 (1991), the disclosure of which is incorporated herein by reference. The term E-cadherin is meant to include recombinant human E-cadherin and recombinant chimeric forms of E-cadherin, which can be prepared using standard recombinant expression methods, or can be purchased commercially (R&D 648-EC-100) .

The term "polypeptide" includes native or artificial proteins, protein fragments, and polypeptide analogs of a protein sequence. A polypeptide can be monomeric or polymeric.

The term "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody that is not associated with naturally-associated components that accompany it in its origin or source of derivation (1) native state, (2) is free from other proteins from the same species, (3) is expressed by a cell from a different species or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or that is synthesized in a cellular system different from the cell from which it naturally arises will be "isolated" from its naturally associated components. A protein can also be made substantially free from naturally associated components by isolation, using protein purification techniques that are well known in the art.

Examples of isolated antibodies include a P-cadherin antibody that has been affinity purified using P-cadherin, and a P-cadherin antibody synthesized by a cell line in vitro.

A protein or polypeptide is "substantially pure", "substantially homogeneous" or "substantially purified" when at least about 60 to 75% of a sample exhibits a single type of polypeptide. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein may typically comprise about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more typically about 95% and may preferably be more than 99% pure. Protein purity or homogeneity can be indicated in a number of ways that are well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band when staining the gel with a general technique in the art known dye. For certain purposes, a higher resolution can be provided by HPLC or other means that are well known in the art for purification.

The term "polypeptide fragment," as used herein, refers to a polypeptide that has an amino terminal and / or carboxy terminal deletion, but wherein the remaining amino acid equivalent is identical to the corresponding positions in the naturally occurring sequence. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids in length. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200 amino acids in length.

The term "analog" or "polypeptide analog" as used herein refers to a polypeptide that comprises a segment that is substantially identical to some reference amino acid sequence and has substantially the same function or activity as the reference amino acid sequence. Polypeptide analogs typically include a conservative amino acid substitution (or insertion or deletion) with respect to the reference sequence. Analogs can be at least 20 or 25 amino acids in length, or can be at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids in length or longer, and can often be as long as the full-length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analog antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions on basis of the germline amino acid sequence. Fragments or analogues of antibodies or immunoglobulin molecules can be easily prepared by those skilled in the art while following the teachings of this disclosure.

In certain embodiments, amino acid substitutions to a β-cadherin antibody or antigen-binding portion thereof are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) bind affinity to modify the formation of protein complexes and (4) confer or modify other physicochemical or functional properties of such ana-logons, but still retain specific binding to β-cadherin. Analogs can include various substitutions to the normally occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions can be generated in the normally occurring sequence, for example, in the portion of the polypeptide outside the domain (s) that form intermolecular contacts. Amino acid substitutions can also be generated in the domain (s) that form intermolecular contacts that can improve the activity of the polypeptide. A conservative amino acid substitution should not substantially alter the structural characteristics of the parent sequence; for example, a replacement amino acid should not alter the antiparallel β plate that forms the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. Usually, glycine and proline should not be used in an anti-parallel beta plate. Examples of technically recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, ed., W.H. Freeman and Company, New York (1984)); Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354: 105 (1991), incorporated herein by reference.

As used herein, the term "antibody" is synonymous with immunoglobulin, and is to be understood as is commonly known in the art. In particular, the term antibody is not limited by any specific method of producing the antibody. For example, the term antibody includes, without limitation, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., β-cadherin) It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody Examples of binding fragments contained within the term "antigen-binding portion" of an antibody include (i) a Fab fragment , a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab1) 2 fragment, a bivalent fragment comprising two Fab fragments connected by means of a disulphide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, ( v) a dAb fragment (Ward et al., Nature, (1989) 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although for the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods through a synthetic linker that allows them to be made as a single protein chain in which the VL and VH regions pair, thereby forming monovalent molecules (known as single-chain Fv (scFv)); see, e.g., Bird et al., Science (1988) 242: 423-426 and Huston et al., Proc. Natl. Acad.

Sci. USA, 85: 5879-5883 (1988)). It is intended that such single-chain antibodies are also contained within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included. Diali-chams are bivalent, bispecific antibodies in which VH and VL domains are expressed in a single polypeptide chain, but using a linker that is too short to allow space between the two domains in the same chain , thereby forcing the domains to pair with complementary domains from another chain, and creating two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak et al. al., Structure, 2: 1121-1123 (1994)).

Still further, an antibody or antigen-binding portion thereof may be a part of larger immunoadhesion molecules formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the strep-tavidine core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas, 6: 93-101 (1995)) and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag for making bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31: 1047-1058 (1994)).

Other examples include where one or more CDRs derived from an antibody are either covalently or non-covalently incorporated into a molecule, making it an immunoadhesin that specifically binds to an antigen of interest, such as β-cadherin. In such embodiments, the CDR (s) may be incorporated as a part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be non-covalently incorporated. Antibody portions, such as Fab and F (ab ') 2 fragments, can be prepared from whole antibodies using conventional techniques, such as digestion of whole antibodies with papain or pepsin, respectively. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein.

Where reference is made herein to an "antibody" with respect to the present invention, it is to be understood that an antigen-binding portion thereof may also be used. An antigen-binding portion competes with the intact antibody for specific binding. See Fundamental Immunology in general, Η. 7 (Paul, W., ed., 2e ed., Raven Press, N.Y. (1989)) 15 (incorporated by reference in its entirety for all purposes). Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, anti-binding portions include Fab, Fab ', F (ab') 2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies diabodies and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. In embodiments with one or more binding sites, the binding sites may be identical to each other or may be different.

As used herein, the term "human antibody" means any antibody in which the variable and constant domain sequences are human sequences. The term includes antibodies with sequences derived from human genes, but which have been altered, e.g., reducing potential immunogenicity, increasing affinity, removing cysteines that could cause undesirable folding, etc. The term includes also such antibodies that are recombinantly produced in non-human cells, which could provide giro-cosylation that is not characteristic of human cells. These antibodies can be prepared in a variety of ways, as described below.

The term "chimeric antibody," as used herein, means an antibody that includes regions derived from two or more different antibodies, including antibodies from different species. For example, one or more of the CDRs of a chimeric antibody may be derived from a human β-cadherin antibody. In one example, the CDRs derived from a human antibody can be combined with CDRs derived from a non-human antibody, such as mouse or rat. In another example, all the CDRs can be derived from human β-cadherin antibodies. In another example, the CDRs derived from more than one human β-cadherin antibody can be combined in a chimeric antibody. For example, a chimeric antibody may be a CDR1 derived from the light chain of a first human β-cadherin antibody, a CDR2 derived from the light chain of a second human β-cadherin antibody, and a CDR3 derived from the light ke-25. include a third human β-cadherin antibody, and CDRs derived from the heavy chain may be derived from one or more other β-cadherin antibodies. Furthermore, the framework regions can be derived from one of the β-cadherin antibodies from which one or more of the CDRs are taken, or from one or more different human antibodies. Furthermore, the term "chimeric antibody" is meant to include any of the combinations mentioned above where the combinations of human and non-human antibodies were carried.

As used herein, the term "humanized antibody" refers to antibodies of non-human origin, wherein the amino acid residues characteristic of antibody sequences of the non-human species are replaced by residues found at the corresponding positions of human antibodies . This "humanization" process is thought to reduce the immunogenicity of the resulting antibody in humans. It will be recognized that antibodies of non-human origin can be humanized using techniques well known in the art. See, e.g., Winter et al., Immunol. Today, 14: 43-46 (1993). The antibody of interest can be manipulated by recombinant DNA techniques so that the CH1, CH2, CH3, hinge domains, and / or the framework domain are substituted with the corresponding human sequence. See, e.g., WO 92/02190 and U.S. Patents 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350 and 5,777,085.

The term "humanized antibody," as used herein, includes chimeric human antibodies and CDR-grafted antibodies within its meaning. Chimeric human antibodies of the invention include the VH and VL of an antibody of a non-human species and the CH and CL domains of a human antibody. The CDR-transplanted antibodies of the invention stem from the replacement of CDRs of the VH and VL of a human antibody with those of the VH and VL of an antibody of an animal other than a human, respectively.

As used herein, the term "ELISA" refers to an assay with an enzyme-linked immunoabsorbent. This determination is well known to those skilled in the art. Examples of this assay can be found in both Vaughan, T.J. et al., Nat. Biotech. , 14: 309-314 (1996), as in

Example 7 of the present application.

The term "surface plasmon resonance" as used herein refers to an optical phenomenon that leaves room for the analysis of biologically specific real-time interactions through the detection of changes in protein concentrations within a biosensor matrix, for example using the BIACORE ™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al., Ann. Biol. Clin. 51: 19-26 (1993); Jonsson et al.; Bio-techniques, 11: 620-627 (1991); Jonsson et al., J. Mol. Recognita 8: 125-131 (1995) and Johnsson et al., Anal. Biochem., 198: 268-277 (1991).

The term "KV refers to the binding affinity equilibrium constant of a specific antibody-antigen interaction. It is said that an antibody specifically binds an antigen when the K" is <1 mM, preferably <100 nM and most preferably <10 nM A KD binding affinity constant can be measured using surface plasmon resonance, for example using the BIACORE ™ system as discussed in Example 6.

The term "Kaf" refers to the dissociation rate constant of a specific antibody-antigen interaction. A Ka 2 dissociation rate constant can be measured using surface plasma resonance, for example using the BIACORE ™ system as discussed in Example 6.

As used herein, the term "P-cadherin-dependent cell adhesion assay" refers to a assay used to measure the ability of a P-cadherin antibody to prevent the adhesion of cells to a receptor P-cadherin that is immobilized on a solid support. This type of determination can be carried out, for example, by immobilizing β-cadherin on a solid support, such as plastic. Cells that overexpress P-cadherin are then allowed to adhere to the solid support via P-cadherin-P-cadherin interactions. The level of adhesion can then be quantified with and without a P-35 cadherin antibody. Adhesion as a function of antibody concentration can then be used to determine an IC50 value. Example 3 provides further 31 details of a P-cadherin-dependent cell adhesion assay that was used to measure IC50 values for P-cadherin antibodies.

As used herein, the term "P-5 cadherin-dependent cell aggregation assay" refers to an assay for measuring the ability of a P-cadherin antibody to prevent the aggregation of cells expressing P-cadherin on their surfaces. For example, this type of assay can use a cell line that overexpresses P-cadherin, placing the cells in suspension and allowing them to form P-cadherin-dependent aggregates. The aggregation assay is then used to quantify the ability of a β-cadherin antibody to prevent this aggregation by measuring the size of cellular aggregates that result with and without the antibody. Cell aggregate size as a function of β-cadherin antibody concentration can then be used to determine an IC50 value. Example 4 provides further details of a P-cadherin-dependent aggregation assay that was used to measure IC 50 values for various P-cadherin antibodies.

As used herein, the term "P-25 cadherin-dependent spherical dislocation assay" refers to an assay for measuring the ability of a P-cadherin antibody to disrupt preformed P-cadherin-dependent cellular aggregations. An IC 50 value can be determined by measuring the size reduction of aggre holes as a function of antibody concentration. Example 5 provides further details of a β-cadherin-dependent spheroid disruption assay that was used to measure teaching values for β-cadherin antibodies.

As used herein, the term "molecular selectivity" refers to the binding affinity of an antibody for a specific antigen that is greater than for a related antigen. For example, the antibodies of the present invention can be selective for β-cadherin over β-cadherin, meaning that the binding affinity of the β-cadherin antibody is at least 2 times greater, e.g. 4 times, or 10 times, or 50 times, or 100 times or more, than for E-cadherin. Such binding affinities can be measured using standard techniques known to those skilled in the art.

The term "epitope" includes any protein determinant that is capable of specific binding to an immunoglobulin or T-cell receptor or to interact with a molecule in any other way. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains, and usually have both specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be "linear" or "conformational". In a linear epitope, all the points of interaction between the protein and the molecule that interacts (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur over amino acid residues in the protein that are separated from each other. Once a desired epitope in an antigen has been determined, it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present invention. Alternatively, during the discovery process, generation 30 and antibody characterization can shed light on information about desirable epitopes. Based on this information, it is then possible to screen competitive antibodies for binding to the same epitope. One approach to accomplish this is to perform cross-competition studies to find antibodies that bind to each other competitively, i.e., the antibodies compete for binding to the antigen. A method with 33 high throughput capacity for "collecting" antibodies, based on their cross-competition, is described in international patent publication WO 03/48731.

The term "competing," as used herein with respect to an antibody, means that a first antibody, or an antigen-binding portion thereof, with a second antibody, or an antigen-binding portion thereof, competes for binding, wherein the binding of the first antibody is detectably lowered with its cognate epitope in the presence of the second antibody, compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably lowered in the presence of the first antibody, may be the case, but this is not necessary. That is, a first antibody can inhibit the binding of a second antibody to its epitope, without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether it is to the same, higher or lesser extent, it is said that the antibodies "cross-compete" with each other to bind their respective epitope (s). Both competing and cross-competing antibodies are included in the present invention. Regardless of the mechanism by which such competition or crosscompetition takes place (e.g., steric prevention, conformational change or binding to a common epitope, or portion thereof, and the like), the average skilled person should recognize, based on the teachings provided herein, that such competing and / or cross-competing antibodies are contained and may be useful for the methods described herein.

As used herein, the term "uses", with reference to a specific gene, means that the amino acid sequence of a specific region in an antibody was ultimately derived from that gene during B cell maturation. For example, the phrase "an amino acid sequence of a heavy chain variable region using a human V" -3 family gene "refers to the situation where the VH region of the antibody was derived from the VH-3 family of gene segments during B cell maturation. In human B cells there are more than 30 distinct, functional variable heavy chain genes with which 10 antibodies are generated. The use of a specifically variable heavy chain gene is therefore characteristic of a preferred antibody-antigen binding binding motif with regard to the combined properties of antigen binding and functional activity. As will be recognized, gene use analysis provides only a limited overview of antibody structure. Since human B cells stochastically generate V-D-J heavy or V-J kappa light chain transcripts, there are a number of secondary processes that take place, including, without limitation, somatic hypermutation, n additions and CDR3 extensions. See, for example, Mendez et al., Nature Genetics 15: 146-156 (1997).

As used herein, the twenty conventional amino acids and their abbreviations follow conventional use. See Immunology - A Synthesis (2nd edition, E.S. Golub and D. R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference.

The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of one of both types of nucleotide. The term includes a few double-stranded forms.

The term "isolated polynucleotide," as used herein, means a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, which, by virtue of its origin, the "isolated polynucleotide" ( 1) is not associated with all or part of polynucleotides with which the "isolated polynucleotide" is found in nature, (2) is operatively linked to a polynucleotide with which it is not connected in nature, or (3) is not occurs in nature as part of a larger sequence.

The term "naturally occurring nucleotides," as used herein, includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides," as used herein, includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotide linkages such as a phosphorothioate, phosphorodithioate, phosphor selenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladate, phosphoramidate and the like. See, e.g., LaPlanche et al., Nucl. Acids Res., 14: 9081 (1986); Stee et al., J. Am. Chein. Soc. 106: 6077 (1984);

Stein et al., Nucl. Acids Res., 16: 3209 (1988); Zon et al., Anti-Cancer Drug Design, 6: 539 (1991); Zon et al., 20 Oligonucleotides and Analogues: A Practical Approach, p. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford,

England (1991)); U.S. Patent 5,151,510; Uhlmann and Peyman, Chemical Reviews, 90: 543 (1990), the descriptions of which are incorporated herein by reference. An oligonucleotide can include a marker for detection, if desired.

"Operatively linked" sequences include both expression control sequences that border on the gene of interest, and expression control sequences that act in trans or remote, thereby controlling the gene of interest.

The term "expression control sequence," as used herein, means polynucleotide sequences that are necessary for effecting the expression and processing of coding sequences with which they are associated. Expression regulation sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e.

Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such regulatory sequences differs depending on the host organism; in prokaryotes, such regulatory sequences usually include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, such regulatory sequences usually include promoters and transcription termination sequence. The term "regulatory sequences" is meant to include at least all components whose presence is essential for expression and processing, and this term may also include additional components whose presence is beneficial, for example, leader sequences and fusion partner sequences.

The term "vector," as used herein, means a nucleic acid molecule that is capable of transporting another nucleic acid to which it is attached. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It is to be understood that "recombinant host cell" and "host cell" mean not only the specific subject cell, but also the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may in fact not be identical to the parent cell, but this progeny is nevertheless contained within the scope of the term "host cell," as herein used.

As used herein, the term "germline" refers to the nucleotide sequences of the antibody genes and gene segments as they are passed from parents to progeny through the germ cells. This germline sequence is distinguished from the nucleotide sequences encoding antibodies in mature B cells that have been altered by recombination and hypermutation events during the course of B cell maturation.

The term "percent sequence being identical" in the context of nucleic acid sequences, means the residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence-identity comparison can be a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least 30 about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. A number of different algorithms are known in the art that can be used to measure nucleotide sequence to be identical. For example, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs 38 in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, for example, the programs FASTA2 and FASTA3, provides alignment and percent sequence-identical-5 of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol., 183: 63-98 (1990); Pearson, Methods Mol. Biol., 132: 185-219 (2000); Pearson, Methods Enzymol., 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71-84 ( 1998), here incorporated by reference 10). Unless specified otherwise, default parameters are used for a specific program or algorithm. The percentage of sequence identity between nucleic acid sequences can for example be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix), or using Gap with its definition parameters such as provided in GCG Version 6.1, incorporated herein by reference.

A reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, it is to be understood that a reference to a specific sequence nucleic acid comprises its complementary strand, with its complementary sequence.

The term "substantial similarity" or "substantial sequence similarity" when referring to a nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand) ), there are nucleotide sequence-identical in terms of at least about 85%, preferably at least about 90% and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known sequence-identical algorithm, such as FASTA, BLAST or Gap, as discussed above.

The term "percent sequence being identical" in the context of amino acid sequences, means the residues in 39 two sequences that are the same when aligned for maximum correspondence. The length of the sequence-identity comparison can be a stretch of at least about five amino acids, usually at least about 20 amino acids, more usually at least about 30 amino acids, typically at least about 50 amino acids, more typically at least about 100 amino acids and even more typically about 150, 200 or more amino acids. A number of different algorithms are known in the art that can be used to measure amino acid sequence identity. For example, amino acid sequences can be compared using FASTA, Gap or Best fit, which are programs in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin.

As applied to polypeptides, the term "substantially identical" or "substantial similarity" means that two amino acid sequences, when optimally aligned, such as using the GAP or BEST-20 FIT programs using default spacing weights as supplied with the programs are at least 70%, sequence 5% or 80% sequence identical, preferably at least 90% or 95% sequence identical, and more preferably at least 97%, 98% or 99% sequence-identical are identical parts. In certain embodiments, residue positions that are not identical differ due to conservative amino acid substitutions.

As the term is used herein, a "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue that has a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Usually, a conservative amino acid substitution will not significantly change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, Me-5 thods Mol. Biol. 243: 307-31 (1994). Examples of groups of amino acids that they have chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; - 5) basic side chains: lysine, arginine and histidine; 6) acid side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substitution groups can be, for example: valine-leucine isoleucine, phenylalanine tyrosine, lysine arginine, alanine valine, glutamate aspartate and asparagine glutamine.

Alternatively, a conservative replacement is any change with a positive value in the PAM250 log probability matrix described in Gonnet et al., Science 256: 1443-45 (1992), incorporated herein by reference. A "moderately conservative" replacement is any change with a non-negative value in the PAM250-25 log probability matrix.

Sequence identity for polypeptides is typically measured using sequence analysis software. Protein analysis software aligns sequences using similarity units of 30 that are assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit" that can be used with default parameters, as specified by the programs, to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from 41 different species of organisms, or between a wild-type protein and an analogue thereof. See, e.g., GCG Version 6.1 (University of Wisconsin, WI). Polypeptide sequences can also be compared using FASTA using default or recommended parameters, see GCG Version 6.1. FASTA (e.g. FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzy-10 mol., 183: 63-98 (1990 Pearson, Methods Mol. Biol. 132: 185-219 (2000)). Another preferred algorithm, when comparing a sequence of the invention with a database containing a large number of sequences from different organisms, is the computer program BLAST, in particular blastp or tblastn, using default parameters as supplied with the programs. See, e.g., Altschul et al., J. Mol. Biol., 215: 403-410 (1990); Altschul et al., Nucleic Acids Res., 25: 3389-402 (1997).

The length of polypeptide sequences compared for homology will usually be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database that contains sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

As used herein, the terms "label" or "labeled" refer to the incorporation of another molecule into the antibody. In one embodiment, the marker is a detectable marker, e.g., the incorporation of a radiolabeled amino acid or the attachment to a polypeptide of biotinyl residues that can be detected by labeled avidin (e.g., streptavidin having a fluorescent marker or enzymatic activity). which can be detected by optical methods. In another embodiment, the label or label may be therapeutic, e.g., a drug conjugate or toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptides labels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 1SN, 35S, 90Y, 99Tc, 113In, 125I, 131I), fluorescent labels (e.g., FITC, rho-10 damine, lanthanide phosphors), enzymatic labels (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucinerite pair) sequences, secondary antibody binding sites, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vin-cristine vinblastine, colchicine, doxorubicin, daunorabicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetraca ine, lidocaine, propranolol, and puromycin and analogs or homologous thereof. In some embodiments, markers are attached by means of mutually spacer arms of different lengths, thereby reducing possible steric prevention.

"Therapeutically effective amount" refers to that amount of the therapeutic agent being administered that will somewhat alleviate one or more of the symptoms of the disorder being treated. With regard to the treatment of cancer, a therapeutically effective amount refers to that amount that has at least one of the following effects: reducing the size of the tumor; inhibiting (i.e., slightly delaying, preferably stopping) 43 tumor metastasis; somewhat inhibiting (i.e., slightly delaying, preferably stopping) tumor growth, and slightly alleviating (or preferably dispelling) one or more symptoms associated with the cancer.

"Treating", "treating" and "treatment" refer to a method for alleviating or eliminating a biological disorder and / or its accompanying symptoms. With regard to cancer, these terms simply mean that the life expectancy of an individual affected by a cancer will be increased, or that one or more of the symptoms of the disease will be reduced.

"Contacting" refers to bringing together an antibody or antigen-binding portion thereof of the present invention and a target β-cadherin, or epitope thereof, such that it. antibody can affect the biological activity of the β-cadherin. Such "contacting" can be accomplished "in vitro", i.e., in a test tube, a petri dish or the like. In a test tube, contacting may only involve an antibody or antigen-binding portion thereof and β-cadherin or epitope thereof, or it may involve whole cells. Cells can also be maintained or grown in cell culture dishes and contacted with antibodies or antigen-binding portions thereof in that environment. In this context, the ability of a specific antibody or antigen-binding portion thereof to influence a β-cadherin-related disorder, i.e., the IC50 of the antibody, can be determined before attempting to use the antibody in vivo with more complex living organisms. For cells outside the organism, there are multiple methods, and these are well known to those skilled in the art, for contacting β-cadherin with the antibodies or antigen-binding portions thereof.

"Abnormal cell growth," as used herein, unless otherwise indicated, refers to cell growth that occurs independently of normal regulatory mechanisms (e.g., loss of contact inhibition), including the abnormal growth of normal cells and the growth of abnormal cells. This includes, but is not limited to, the abnormal growth of: tumor cells (tumors) that multiply rapidly by expressing a mutated tyrosine kinase or the overexpression of a receptor tyrosine kinase; benign and malignant cells from other proliferative diseases where abnormal tyrosine kinase activation occurs; any tumors that rapidly replicate by receptor tyrosine kinases; any tumors that are rapidly multiplying by abnormal serine / threonine kinase activation; benign and malignant cells from other proliferative diseases where abnormal serine / threonine kinase activation occurs; tumors, both benign and malignant, that express an activated Ras-20 oncogene; tumor cells, both benign and malignant, where the Ras protein is activated due to oncogenic mutation in another gene; benign and malignant cells from other proliferative diseases in which abnormal Ras activation takes place. Examples of such benign proliferative diseases are psoriasis, benign prostatic hypertrophy, human papillomavirus (HPV) and restinosis. "Abnormal cell growth" also refers to, and includes, the abnormal growth of cells, both benign and malignant, that results from the activity of the enzyme farnesyl protein transferase.

The terms "abnormal cell growth" and "hyperproliterative disorder" are used interchangeably in this application.

"In vitro" refers to procedures performed in an artificial environment, such as, for example, without limitation, in a test tube or culture medium.

"In vivo" refers to procedures performed within a living organism, such as, without limitation, a mouse, rat or rabbit.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid and nucleic acid sequences of SEQ ID NOs: 1-347.

DETAILED DESCRIPTION OF THE INVENTION Human β-cadherin antibodies

This invention relates to isolated human 10 antibodies, or antigen-binding portions thereof, that bind to human β-cadherin. Preferably, the human antibodies are recombinant human β-cadherin antibodies that have a greater affinity for β-cadherin than for E-cadherin. Various aspects of the invention relate to both such antibodies and antigen-binding portions, and pharmaceutical compositions thereof, as well as to nucleic acids, recombinant expression vectors, and host cells for making such antibodies and antigen-binding portions. Methods for using the antibodies and antigen-binding portions of the present invention to detect human β-cadherin or to inhibit human β-cadherin activity, whether in vitro or in vivo, are also encompassed by the invention contains.

The β-cadherin amino acid and nucleotide sequences from various species, including humans, are known (see, e.g., registration number NM_001793.3). Human β-cadherin, or antigenic portions thereof, can be prepared by methods well known to those skilled in the art, or can be purchased from commercial vendors (e.g., R&D Systems 861-PC-100).

In certain embodiments, antibodies of the present invention are IgGs, designated: 194-e06; 35 194-a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b 9; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-

AC

e06; 129-1c4 and g-129-1c4. As discussed in more detail in Example 1, high throughput screening of a scFv phage display library was used to identify the 129-lc4 scFv, which was then converted to an IgG. 129-1c4 represents the guideline [lead] antibody identified during the first phage display screening, and is the parent antibody from a lineage from which several other antibodies of the present invention were derived. Several of such derived antibodies are referred to as 194-e06; 194-a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eOl; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09 and 200-h06, and represent optimized antibodies within the 129-lc4 parent descent. The g-129-lc4-15 antibody is the germ-version of the 129-lc4 parent antibody, with certain amino acids in the frame regions of the VH and VL domains mutated, matching them with those in the germline frame regions. Any of the aforementioned antibodies may also be germinated so that the framework region sequences are identical to the germline framework sequences as in g-129-lc4. In one embodiment of the present invention, the antibodies are g-194-b09, g-194-g09, g-196-g03, g-196-h02, g-194-e01, g-195-ell, g-200- h06 and g-194-e06 for example the germinated versions of 194-b09, 194-g09, 196-g03, 196-h02, 194-eO1, 195-ell, 200-h06 and 194-e06, respectively. The specific amino acids that were mutated to arrive at the germinated versions are clear to those skilled in the art from comparing the sequences of a germinated vs.. a non-germinated antibody. As discussed below, specific amino acid sequences of the antibodies of the present invention are described in Tables 1-3 and Figure 1.

Antibodies of the present invention were generated with a strong preference for the use of the VH3 gene family of heavy chain variable regions.

47

In particular, the 129-lc4 parent antibody derived from the variable VH3-23 gene segment. In human B cells, there are more than 30 distinct, functional, variable heavy chain genes with which antibodies are generated. Preference is therefore characteristic of a preferred antibody-antigen binding binding motif with regard to the combined properties of antigen binding and functional activity.

As will be recognized, gene use analysis 10 provides only a limited overview of antibody structure. Since human B cells stochastically generate V-D-J heavy or V-J kappa light chain transcripts, a number of secondary processes are taking place, including, without limitation, somatic hypermutation, n additions and CDR3 extensions. See, for example, Mendez et al., Nature Genetics 15: 146-156 (1997) and US published patent application 2003-0070185, filed February 19, 2002. Accordingly, to further investigate antibody structure of the present invention, predicted amino acid sequences of the antibodies were provided. generated from the cDNA1s obtained from the clones. In addition, N-terminal amino acid sequences were obtained by protein sequencing. Figure 1 provides nucleotide and amino acid sequences of the variable regions of the heavy and light chain of several of the antibodies of the present invention.

Each of the specific antibodies mentioned above can be described by its variable do-mein sequences of the heavy [VH] and light (V L) chains, as indicated in Tables 1 and 2. The specific sequences to which is referred to these SEQ ID NOs, shown in Figure 1. As indicated in Tables 1 and 2, the corresponding VH and VL-35 amino acid and DNA sequences of the antibodies mentioned above are described. by SEQ ID NOs: 1-23, 68-90 and 320-343.

48

Table 1 - Human P-cadherin antibodies

Anti-Segmental identifier (SEQ ID NO :) body VB Vh

Amino acid DNA - amino acid - DNA

-E06__1__68__14__81 a02__2__69__14__81 194 194- 194 195- -b09__2__69__15__82 ell__3__70__16__83 194-g09__4__71__17__84 -h02__4__71__23__90 196 194- 196- eOl__5__72__18__85 dlO__6__73__23__90 g03__7__74__23__90-196 196-e06__8__75__23__90 195- a09__9__76__23__90 a09__10__77__19__86-198 200-h06__11__78__20__87 129-1C4__12__79__21 88 5 Table 2 _Gekiemlijnde human P- cadherin antibodies_

Anti-Sequence Identifier (SEQ ID NO :) body V "__

Amino acid DNA Amino acid DNA

g-194 -b09 320__332__326__338 g-194 -g09 321__333__327__339 g-196 -g03 322__334__328__340 g-196 -h02 323__335__329__341 g-194 -eQl 324__336__330__342 g-194 -e06 325__337__321__3__

Additional antibodies and antigen-binding portions of the present invention can also be described as comprising the various CDR and FR sequences that form the heavy and light chain variable regions of the antibodies indicated in Tables 1 and 2. Accordingly. SEQ ID NOs corresponding to different CDR and FR sequences of antibodies of the present invention are indicated in Table 3. Furthermore, numerous randomized mutations in the CDR3 regions of the heavy and light chain of the parent antibody 129-1c4 was performed, yielding improved β-cadherin affinity ranging from a 10-fold to a 417-fold improvement, as measured by an epitope competition assay (see Example 8). The SEQ ID NOs of these mutated VH and VL CDR3 sequences (SEQ ID NOs: 91-256, and 257-319) are also included in Table 3 below.

Table 3 _SEQ ID NOs: __ Description_ _24__VH-CDR1_ _25__VH - CDR2_ _26-37, 91-256__VH-CDR3_ _38__V ^ -CDR1_ _39__VL - CDR2_ _40-47, 257-319__VL-CDR3_ _48__Vh-501_VH-50H_VH-50H_VH-50H_VH-50H_VH-50H_VH-50H_VH-50H_VH-50H_VH FR3_ _56, 57__VH-FR4_ _58, 59__VL-FR1_ _ 60-62__VL-FR2_ __63-66__VL-FR3_ ___ 67_ | __VL - FR4_ 50

Method of producing antibodies

Phage display libraries

The antibodies or antigen-binding portions of the present invention can be prepared according to various methods known in the art. For example, phage splay techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for β-cadherin. These libraries can then be screened to identify and isolate antibodies with the desired affinity for β-cadherin.

Recombinant human β-cadherin antibodies of the present invention can be isolated, for example, by screening a recombinant combinatorial antibody library. Preferably, the library is an scFv phage display library generated using human V L and V H cDNAs prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g. the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the SurfZAP ™ phage display kit from Stratagene, catalog no. 240612). There are also other methods and reagents that can be used in the generation and screening of antibody display libraries (see, e.g., U.S. Patent 5,223,409; PCT publications WO 92/18619, WO 91/17271, WO 92/20791, WO 92 / 15679, WO 93/01288, WO 92/01047 and WO 92/09690; Fuchs 30 et al., Bio / Technology 9: 1370-1372 (1991); Hay et al.,

Hum. Antibod. Hybridomas, 3: 81-85 (1992); Huse et al.

Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol. Biol. 226: 889-896 (1992); Clackson et al., Nature 352: 624-628 (1991); Gram et al.

Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992); Garrad et al., Bio / Technology, 9: 1373-1377 (1991); Hoogenboom et 53.

al., Nuc. Acid Res., 19: 4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); and Griffiths et al., EMBO J., 13: 3245-3260 (1994), all of which are incorporated herein by reference).

Another method for preparing a library of antibodies for use in phage display techniques includes the steps of immunizing a non-human animal comprising loci for human immunoglobulin, with β-cadherin or an antigenic portion thereof, whereby 10 an immune response is created, extracting antibody-producing cells from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the invention from the extracted cells, inversely transcribing the RNA produced by cDNA, amplifying the cDNA using primers, and deploying it in such a way of the cDNA in a phage display vector that antibodies are expressed in the phage. For the production of such repertoires, it is not necessary to immortalize the B cells from the immunized animal. On the contrary, the primary B cells can be used directly as a source of DNA. The mixture of cDNAs obtained from the B cells, eg derived from spleen, is used to prepare an expression library, for example, a phage display library that is transfected into E. coli. Finally, clones are identified from the library that produce binding affinities of a desired size for the antigen, and the DNA encoding the product responsible for such binding is recovered and manipulated for standard recombinant expression. Page display libraries can also be constructed using previously manipulated nucleotide sequences and screened in a similar manner. Typically, cDNAs encoding heavy and light chains are supplied or connected independently, thereby forming Pv analogs for production in the 52 phage library. The phage library is then screened for the antibodies with the highest affinities for β-cadherin, and the genetic material is recovered from the appropriate clone. Further rounds of screening may increase the affinity of the isolated original antibody.

In one embodiment, to isolate and produce human β-cadherin antibodies with the desired characteristics, a human β-cadherin antibody, as described herein, is first used to select human heavy and light chain sequences with similar binding actions. against P-cadherin, using the epitope imprinting methods described in PCT publication WO 93/06213, incorporated herein by reference. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described in PCT publication WO 92/01047, McCafferty et al., Nature, 348: 552-554 (1990); and Griffiths et al., EMBO J. 12: 725-734 (1993), all incorporated herein by reference. The scFv antibody libraries can be screened using human β-cadherin as the antigen. The phage library is screened for the antibodies with the highest affinities for β-cadherin, and the genetic material is recovered from the appropriate clone. Further rounds of screening may increase the affinity of the isolated original antibody.

Once the first human VL and VH domains have been selected, then "mixing and matching" experiments can be performed, screening different pairs of the initially selected Vh and VH segments for P-cadherin binding to select preferred VL / VH pair combinations. These mixing and matching experiments can also be performed after the VH and VL segments have been randomly mutated for optimized binding as described below. Further 53, to further improve the quality of the antibody, the VL and VH segments of the preferred VL / VH pa (s) can be mutated randomly, preferably within the CDR3 region of V H and / or V L, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished, for example, by amplifying VH and VL-10 domains using PCR primers that are complementary to the VH-CDR3 or VL-CDR3, respectively, to which primers "at certain positions" small amount has been added "of any mixture of the four nucleotide bases that the resulting PCR products encode VH and VL segments into which random mutations have been introduced into the VH and / or VL-CDR3 regions. These randomly mutated VH and VL segments can be re-screened for binding to P-cadherin, and sequences that exhibit high affinity and low-rate for P-20 cadherin can be selected. As previously discussed, various VH and VL-CDR3 sequences of the present invention that were randomly mutated and exhibited improved affinity are indicated by SEQ ID NOs: 91-256 and 257-319.

After screening and isolation of a β-cadherin antibody of the invention based on a recombinant immunoglobulin display library, nucleic acids encoding the selected antibody can be recovered from the display package (e.g. from the phage genome) and subcloned into others expression vectors using standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention as described below. To express a recombinant hu-35 antibody isolated by screening from a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a host cell as described below.

Immunization In another embodiment, human β-cadherin antibodies can be produced by immunizing a non-human, transgenic animal that encloses anything, or everything from the human immunoglobulin heavy chain and light chain loco. 10 barrel, with a β-cadherin antigen. The non-human animal may, for example, be a XEN0M0USE ™ animal. (Abgenix, Ine., Fremont, CA).

XENOMOUSE ™ mice are engineered mouse strains that include large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7: 13-21 (1994) and U.S. Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 / 09560 and WO 00/037504.

The methods described in these documents can be modified as described in U.S. Patent 5,994,619, which is incorporated herein by reference. US patent 5,994,619 describes methods for producing new cultured inner cell mass (CICM) cells and cell lines, derived from pigs and cows, and transgenic CICM cells in which heterologous DNA is inserted. Transgenic CICM cells can be used to produce cloned transgenic embryos, fetuses and offspring. The '619 patent also describes methods for producing transgenic animals that are capable of transferring the heterologous DNA to their offspring. Examples of non-human animals that can be used with these methods include rats, sheep, pigs, goats, cattle, chicken, and horses.

XENOMOUSErM mice produce a late human repertoire of fully human antibodies and generate antigen-specific human antibodies. In some embodiments, the XENOMOUSE ™ mice contain about 80% of the human antibody V gene repertoire through the introduction of fragments of megabase size and germ line configuration of the human heavy chain loco and 10 loca for the kappa -light chain in the artificial yeast chromosome [yeast artificial chromosome] (YAC). In other embodiments, XENOMOUS ™ mice further contained substantially all of the human lambda light chain locus. See Mendez et al., Nature Genetics, 15: 146-156 (1997), Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998) and WO 98/24893, the disclosures of which are incorporated herein by reference.

In some embodiments, the non-human animals that include human immunoglobulin genes are animals that have a "minilocus" for a human immunoglobulin. In the minilocus approach, an exogenous Ig locus is simulated by the inclusion of individual genes from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH-25 genes, a mu constant domain, and a second constant domain (preferably a gamma constant domain) are formed into a construct for insertion in an animal. This approach is described in U.S. Patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215 and 5,643,763, incorporated herein by reference.

Non-human animals, as described above, can then be immunized with a β-cadherin antigen, as described below, under conditions that allow antibody production. Antibody-producing cells are isolated from the animals, and nucleic acids encoding the heavy and light chains of the β-cadherin antibody of interest are isolated from the isolated antibody-producing cells or from an immortalized cell line produced from such cells . These nucleic acids are then manipulated using techniques known to those skilled in the art and as further described below, thereby reducing the amount of non-human sequence, i.e., humanizing the antibody, thereby reducing the immune response in humans.

In some embodiments, the β-cadherin antigen may be isolated and / or purified β-cadherin. In some embodiments, the β-cadherin antigen is hu-15 and is β-cadherin. In other embodiments, the β-cadherin antigen may be a cell that expresses or over-expresses β-cadherin. In other embodiments, the β-cadherin antigen is a recombinant protein expressed on the basis of yeast, insect cells, bacteria such as E. coli, or other resources by recombinant technology. The immunization of animals can be done by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: 25 Cold Spring Harbor Press (1990). Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane, supra, and U.S. Patent 5,994,619. For example, the β-cadherin antigen can be administered with an adjuvant, thereby stimulating the immune response. Typical adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyldipeptides) or ISCOM (immunostimulatory complexes). Such adjuvants can protect the polypeptide against rapid dispersion by subjecting it to a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system. Preferably, if a polypeptide is administered, the immunization schedule will involve two or more administrations of the polypeptide, spread over a few weeks.

For example, after immunization of a transgenic animal as described above with β-cadherin, primary cells (e.g., spleen cells or B cells of peripheral blood) can be isolated from the immunized transgenic animal, and individual cells that produce antibodies specific to the desired antigen can be identified. Polyadenylated mRNA from each individual cell is then isolated, and reverse transcription polymerase chain reaction (RT-PCR) is performed using sense primers that fuse with variable region sequences (e.g., degenerate primers that contain the majority, or recognize all of the FR1 regions of the human heavy and light chain variable region genes and antisense primers fusing with sequences for the constant or connecting region. cDNAs from the variable domains of the heavy and light chain are then cloned and expressed in any suitable host cell, eg a myeloma cell, as chimeric antibodies with respective immunoglobulin constant regions, such as the constant domain of the heavy chain and k or λ constant domains. See Babcook et al., Proc. Natl. Acad. Sci. USA, 93: 7843-48, (1996), incorporated herein by reference. P-cadherin antibodies can then be identified and isolated as described herein.

Recombinant methods for producing antibodies

An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody in a recombinant manner, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and heavy chains of the antibody immunoglobulin such that the light and heavy chains are expressed in the host cell and are preferably secreted to the medium in which the host cells are grown, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used for obtaining the heavy and light chain genes of the antibody, for incorporating these genes into recombinant expression vectors, and for introducing the vectors into host cells, such as those being used. -15 wrote in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Cold

Spring Harbor, N.Y. (1989), Ausubel, F.M. et al. (Eds.),

Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Patent No. 4,816,397, the disclosures of which are incorporated herein by reference.

Mutations and modifications

To express the β-cadherin antibodies of the present invention, DNA fragments encoding VH and VL regions can first be obtained using any of the methods described above. Various mutations, deletions and / or additions can also be introduced into the DNA sequences using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods, such as PCR-mediated mutagenesis, the mutated nucleotides being incorporated into the PCR primers such that the PCR product contains the desired mutations, or site-directed mutagenesis. For example, one type of substitution that can be generated is to change one or more cysteines in the antibody, which can be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there may be a substitution of a non-canonic cysteine. The substitution can be generated in a CDR or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonic.

The antibodies may also be mutated in the variable domains of the heavy and / or light chains, e.g., to alter a binding property of the antibody. For example, a mutation can be produced in one or more of the CDR regions for increasing or decreasing the Kj, for the β-cadherin antibody, for increasing or decreasing Kaβ or for altering the binding specificity of the antibody. Techniques in the site-directed mutagenesis are well known in the art. See, e.g., Sambrook et al. And Ausubel et al., Supra, which is incorporated herein by reference. For example, as discussed in more detail in Example 8, numerous alternative VH and VL-CDR3 sequences of the 129-1c4 mother were made according to the procedures discussed above, and are shown in Figure 1 as SEQ ID NOs: 91 to 256 (VH-CDR3 variants) and SEQ ID NOs: 257 to 319 (VL-CDR3 variants).

A mutation can also be generated in a frame region or constant domain, to increase the half-life of a β-cadherin antibody. See, e.g., PCT publication WO 00/09560, incorporated herein by reference. A mutation in a framework region or constant domain can also be generated to change the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to modify such. properties such as complement fixation, FcR binding and antibody-dependent, cell-mediated cytotoxicity [antibody-dependent cell-mediated cytotoxicity] (ADCC). According to the invention, a single antibody may have mutations 60 in one or more of the CDRs or framework regions of the variable domain or in the constant domain.

In a process known as "germline", certain amino acids can be mutated in the VH and VL sequences to match those naturally found in germline VH and VL sequences. In particular, the amino acid sequences of the framework regions in the V H and V L sequences can be mutated to match the germline sequences, thereby reducing the risk of immunogenicity when the antibody is administered. Germline DNA sequences for human VH and VL genes are known in the art (see e.g. the humane germline sequence database "Vbase"; see also Kabat, EA et al. (1991) Sequences of Proteins of Immunological Inte -15 rest, Fifth Edition, US Department of Health and Human Services, NIH Publication 91-3242, Tomlinson et al. (1992) J. Mol. Biol. 227: 776-798 and Cox et al., E. J. Immunol. 24: 827-836 (1994); the content of each of which is explicitly incorporated herein by reference).

Another type of amino acid substitution that can be generated is the removal of potential proteolytic sites in the antibody. Such sites may occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is the removal of asparagine-glycine pairs, which form potential deamidation sites, by altering one or both of the residues. In another example, the C-terminal lysine can be cleaved from the heavy chain of a β-cadherin antibody of the invention. In various embodiments of the invention, the heavy and light chains of the β-cadherin antibodies may optionally comprise an N-terminal signal sequence, such as those indicated by SEQ ID NOs: 346 and 347.

Once DNA fragments encoding the VH and VL segments of the present invention have been obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example for converting the variable genes region to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a DNA fragment coding for V L or V H is functionally linked to another DNA fragment coding for another protein, such as a constant region of an antibody or a flexible linker. The term "operably linked", as used in this context, is intended to mean that the two DNA fragments are linked and such that the amino acid sequences encoded by the two DNA fragments remain in phase.

The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding constant regions of the heavy chain (CH1, CH2 and CH3). The sequences of genes for the constant region of the human heavy chain are known in the art (see, e.g., Rabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., US Department of Health and Human Services, NIH Publication 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The constant region of the heavy chain can be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but is most preferably a constant region of IgG1 or IgG2. The sequence for the constant region of IgG1 can be one of the different alleles or allotypes known to occur among different individuals, such as Gm (1), Gm (2), Gm (3) 62 and Gm (17). These allotypes represent naturally occurring amino acid substitutions in the constant regions of IgG1. For example, the constant region of the heavy chain of IgG1 can be SEQ ID NO: 344. For a heavy chain gene of a Fab fragment, the V1 coding DNA may be operably linked to another DNA molecule that only encodes the CH1 constant region of the heavy chain. The constant region of the heavy chain CH1 can be derived from one of the genes for the heavy chain.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to a another DNA molecule that encodes the constant region of the light chain, CL. The sequences of genes for the human light chain constant region are known in the art (see, e.g., Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., US Department of Health and Human Services, NIH publication 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The constant region of the light chain can be a kappa or lamb-25 da constant region. The kappa constant region can be one of the different alleles known to occur among different individuals, such as

Inv (1), Inv (2) and Inv (3). The lambda constant region can be derived from one of the three lambda genes. The constant region of the light chain of IgG1 can be, for example, SEQ ID NO: 347.

To create a scFv gene, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, e.g. encoding the amino acid sequence (Gly4-Ser) 3 that the VH and Vb sequences can be expressed as a contiguous single chain protein, with the VL and VH regions linked by the flexible linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl.

Acad. Sci. USA 85: 5879-5883 (1988); McCafferty et al., Nature, 348: 552-554 (1990)). The single chain antibody can be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyva-10 spring antibodies can be generated that specifically bind to β-cadherin and to another molecule.

In another embodiment, a fusion antibody or immunoadhesin can be made that comprises all or a portion of a β-cadherin antibody of the invention, linked to another polypeptide. In another embodiment, only the variable domains of the β-cadherin antibody are linked to the polypeptide. In another embodiment, the V-domain of a P-cadherin antibody is linked to a first polypeptide, while the VL-domain of a P-cadherin antibody is linked to a second polypeptide that associates with the first polypeptide in such a way that the VH and VL domains can interact with each other, thereby forming an antigen-binding site. In another preferred embodiment, the VH domain is separated from the V L domain by means of a linker that the V H and V L domains can interact with each other. The V H linker-VL antibody is then linked to the polypeptide of interest. In addition, fusion antibodies can be created where two (or more) single-chain antibodies are linked together. This is useful if one wants to create a divalent or polyvalent antibody in a single polypeptide chain, or if one wants to create a bispecific antibody.

In other embodiments, other modified antibodies can be prepared using 64 β-cadherin antibody-encoding nucleic acid molecules. "Kappa bodies" ["Kappa bodies"] (111 et al., Protein Eng. 10: 949-57 (1997)), "Mini bodies" ["Minibodies"] (Martin et al., EMBO J., 13: 5303-9 (1994)), "Diabodies" ["Diabodies"] (Holliger et al., Proc. Natl. Acad. Sci.

USA, 90: 6444-6448 (1993) or "Janusinen" ["Janusins"] (Traunecker et al., EMBO J., 10: 3655-3659 (1991) and Traucecker et al., Int. J. Cancer, (Suppl.) 7: 51-52 (1992)) can be prepared, for example, using standard molecular biology techniques while following the teachings of the description.

Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods, including fusion of hybrids or joining Fab 'fragments. See, e.g., Songsi-vilai & Lachmann, Clin. Exp. Iimunol. , 79: 315-321 (1990),

Kostelny et al., J. Iimunol., 148: 1547-1553 (1992). In addition, bispecific antibodies can be formed as "diabodies" or "Janusines". In some embodiments, the bispecific antibody binds to two different epitopes of β-cadherin. In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions derived from a human β-cadherin antibody provided herein.

Vectors and host cells

To express the antibodies and antigen binding portions of the invention, DNAs encoding partial light and heavy chains or light and full length heavy chains obtained as described above are deployed in expression vectors such that the genes become functional associated with transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational regulatory sequences within the vector fulfill their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. Express vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes and the like. The antibody gene is ligated into a vector such that transcriptional and translational regulatory sequences within the vector fulfill their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be used in separate vectors. In a preferred embodiment, both genes are used in the same expression vector. The antibody genes are inserted into the expression vector using standard methods (e.g., ligation of complementary restriction sites in the antibody gene fragment and the vector, or ligation of blunt end if no restriction sites are present).

A suitable vector is one that encodes a functionally complete sequence for human immunoglobulin CH or -CL, with appropriate restriction sites engineered so that any V H or V L sequence can be easily deployed and expressed, such as described above. With such vectors, splicing usually takes place between the splice donor site in the deployed J region and the splice acceptor site that precedes the human C domain, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination take place at native chromosomal sites, downstream of the coding regions. The recombinant expression vector can also encode a signal peptide r u u that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is connected in phase to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that regulate the expression of the antibody chain genes in a host cell. It will be recognized by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the desired level of expression of protein, and so on. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g. the major late promoter of adenovirus [adenovirus major late promoter] (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For a further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Patent 5,168,062, U.S. Patent 4,510,245 and U.S. Patent 4,968,615. Methods for both the expression of antibodies in plants, including a description of promoters and vectors, and the transformation of plants are known in the art. See, e.g., U.S. Patent No. 6,517,529, incorporated herein by reference. Methods for expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art.

67

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (e.g., origins of replication), and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference). For example, the selectable marker gene typically confers drug resistance, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in methotrexate selection / amplification dhfr host cells), the neomycin phosphotransferase gene (for G418 selection) and the glutamate synthetase gene .

Nucleic acid molecules encoding β-cadherin antibodies and vectors comprising these nucleic acid molecules can be used for the transfection of a suitable mammalian, plant, bacterial or yeast host cell. The transformation can be carried out using any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextrange-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) into liposomes and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells through viral vectors. Methods for transforming cells are well known in the art. See, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455, incorporated herein by reference. Methods for transforming 68 plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, for example, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, hamster kidney [baby hamster kidney] (BHK) cells, kidney cells of African green meerkat 15 (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to leave room for the antibody's expression in the host cells or, more preferably, secretion of the antibody to the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Ara-bidopsis, duckweed, corn, wheat, potato, and so on. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Furthermore, the expression of antibodies of the invention based on production cell lines can be enhanced 69 using a number of known techniques. For example, the glutamine synthetase (the GS system) and DHFR gene expression systems are common approaches to enhancing expression under certain conditions. Highly expressed cell clones can be identified using conventional techniques such as limited dilution cloning and Microdrop technology. The GS system is discussed in European patents 0 216 846, 0 256 055, 0 10 323 997 and 0 338 841.

Antibodies expressed by different cell lines or in transgenic animals are likely to have a different glycosylation. However, all antibodies encoded by the nucleic acid molecules provided herein, or which include the amino acid sequences provided herein, are part of the present invention, regardless of the glycosylation of the antibodies.

Transgenic animals and plants β-cadherin antibodies of the invention can also be produced transgenically by the generation of a mammal or plant that is transgenic to the sequences of interest for the heavy and light chain of an immunoglobulin, and the production of the anti-25 body in a form that can be extracted from it. In connection with transgenic production in mammals, β-cadherin antibodies can be produced in, and extracted from, the milk of goats, cows or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference. In some embodiments, non-human transgenic animals comprising human immunoglobulin locus are immunized with β-cadherin or an immunogenic portion thereof, as described above. Methods for making antibodies in plants are described, for example, in U.S. Patents 6,046,037 and 5,959,177, incorporated herein by reference.

7 0

In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding a P-cadherin antibody, or antigen-binding portion thereof, of the invention, into the animal or plant using of conventional transgenic techniques. See Hogan and U.S. Patent 6,417,429, supra. The transgenic cells used to make the transgenic animal may be embryonic stem cells or somatic cells, or a fertilized egg. The transgenic non-human organisms can be chimeric, non-chimeric heterozygotes, and non-chimeric homozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, 2 * ed., Cold 15 Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (20 00); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999), all incorporated herein by reference. In some embodiments, the transgenic non-human animals have targeted disruption and replacement with a targeting construct that encodes a heavy chain and / or a light chain of interest. The β-cadherin antibodies can be made in any transgenic animal. In a preferred embodiment, the non-human animals are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal expresses those encoded polypeptides in blood, milk, urine, saliva, tears, mucus, and other bodily fluids.

30 Class changeover

The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass (e.g., IgG1, IgG2, IgG3, or IgG4) of β-cadherin antibodies can be determined by any method known in the art. Typically, the class and subclass of an antibody can be determined using antibodies specific for a specific class and subclass of 71 antibody. Such antibodies are commercially available. The class and subclass can be determined using either ELISA, or Western Blot, as well as other techniques. Alternatively, class 5 and subclass may be determined by sequencing all or part of the constant domains of the heavy and / or light chains of the antibodies, comparing their amino acid sequences with the known amino acid sequences of different classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies. The β-cadherin antibodies of the present invention can be an IgG, an IgM, an IgE, an IgA or an IgD molecule. The β-cadherin antibodies may, for example, be an IgG that is an IgG1, IgG2, IgG3 or an IgG4 subclass. In one embodiment, the β-cadherin antibodies may have a constant region of the heavy chain indicated by SEQ ID NO: 344, and a constant region of the light chain indicated by SEQ ID NO: 345.

One aspect of the invention provides a method for converting the class or subclass of a β-cadherin antibody to another class or subclass. In some embodiments, a nucleic acid molecule encoding a V L or V H that does not include sequences encoding CL or CH is isolated using methods well known in the art. The nucleic acid molecule is then operably linked to a nucleic acid sequence that encodes a CL or CH derived from a desired immunoglobulin class or subclass. This can be accomplished using a vector or nucleic acid molecule that comprises a CL or CH chain as described above. A β-cadherin antibody that was originally, for example, IgM, can be switched in class to an IgG. Furthermore, the class switch can be used to convert one IgG subclass to another, e.g. from IgG1 to IgG2. Another method of producing an antibody of the invention comprising a desired isotype comprises the steps of isolating a nucleic acid encoding a heavy chain of a β-cadherin antibody and a nucleic acid encoding a light chain of a β-cadherin antibody, isolating the sequence coding for the VH region, ligating the VH sequence with a sequence coding for a constant domain of the heavy chain of the desired isotype, expressing the light chain gene 10 and the heavy chain construct in a cell, and collecting the β-cadherin antibody with the desired isotype.

De-immunized antibodies

In another aspect of the invention, the antibodies or antigen-binding portions thereof may be deimmunized to reduce their immunogenicity, using the techniques described in, e.g., PCT publications WO98 / 52976 and WO00 / 34317 (here included by reference).

Derivatized and labeled antibodies

A β-cadherin antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). Usually, the antibodies or the portion thereof are derivatized such that the β-cadherin binding is not adversely affected by the derivatization or labeling. Accordingly, it is intended that the antibodies and antibody portions of the invention comprise both intact and modified forms of the human β-cadherin antibodies described herein. For example, an antibody or antibody portion of the invention may be operably linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g. a bispecific antibody or diabody), a detection agent, a marker, a cytotoxic agent, a pharmaceutical agent, 73 and / or a protein or peptide that associates the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine nucleus) call) can mediate.

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, eg for creating bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, with two differently reactive groups that are separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobi-functional (e.g., disuccinimidylsuberate). Such couplers are available from Pierce Chemical Company, Rockford, IL.

Another type of derivatized antibody is a labeled antibody. Useful detection agents with which an antibody or antigen-binding portion of the invention can be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalene sulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. An antibody can also be labeled with enzymes useful for detection, such as horseradish telperoxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labeled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be observed. For example, when the horseradish peroxidase agent is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is detectable. An antibody can also be labeled with biotin, and detected by the indirect measurement of avidin or streptavidin binding. An antibody can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope labels). In some embodiments, markers 5 are attached by means of mutually spacer arms of different lengths, thereby reducing possible steric prevention. A β-cadherin antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups are useful for improving the biological characteristics of the antibody, e.g., for increasing the half-life in serum.

Binding affinity of P-cadherin antibodies for P-15 cadherin

The binding affinity (KD) and the dissociation rate (Kaf) of a P-cadherin antibody or antigen-binding portion thereof for P-cadherin can be determined by methods known in the art. The binding affinity can be measured using ELISAs, RIAs, flow cytometry or surface plasma monononance, such as BIACORE ™. The dissociation rate can be measured using surface plasmon resonance. Preferably, the binding affinity and the dissociation rate are measured using surface plasma monononance. More preferably, the binding affinity and the dissociation rate are measured using BIACORE ™. One can determine whether an antibody has substantially the same KD as a β-cadherin antibody by methods known in the art. Such methods for determining Κρ and Kaf can be used during both the first screening stage and during subsequent optimization stages.

Identification of β-cadherin epitopes recognized by β-cadherin antibodies

The invention provides human β-cadherin antibodies that bind to β-cadherin and compete or cross-compete with and / or bind the same epitope as one of the antibodies described in Tables 1 or 2. One can determine whether an antibody binds to the same epitope or cross-competes with a P-5 cadherin antibody of the present invention to bind by methods known in the art. In one embodiment, the β-cadherin antibody of the invention is allowed to bind to β-cadherin under saturating conditions, and then the ability of the test antibody to bind to β-cadherin is measured. If the test antibody is able to bind to P-cadherin at the same time as the P-cadherin antibody, then the test antibody binds to a different epitope than the P-cadherin antibody. However, if the test antibody is unable to bind to P-cadherin at the same time, the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the human P -cadherin antibody. This experiment can be performed using ELISA, RIA, BIACORE ™ or flow cytometry. In a preferred embodiment, the experiment is performed using ELISA.

Inhibition of P-cadherin activity by a P-cadherin antibody P-cadherin antibodies that inhibit P-cadherin activity can be identified using a number of assays. For example, a cell aggregation assay provides a method for measuring β-cadherin-dependent cellular aggregation. This type of assay uses a cell line that over-expresses P-cadherin, placing the cells in suspension and allowing them to form P-cadherin-dependent aggregates. The aggregation assay is then used to quantify the ability of a β-cadherin antibody to prevent this aggregation by measuring the size of resulting cellular aggregates with and without the antibody. Cell aggregate size as a function of β-cadherin antibody concentration can then be used to determine an IC50 value. Example 4 provides further details of a P-cadherin-dependent aggregation assay that was used to measure teaching values for various P-cadherin antibodies.

Cell adhesion assays can also be used to measure the ability of a P-cadherin antibody to prevent the adhesion of cells to a receptor P-cadherin immobilized on a solid support. This type of determination can be carried out, for example, by immobilizing β-cadherin on a solid support, such as plastic. Cells that overexpress P-cadherin are then allowed to adhere to the solid support via P-cadherin-P-cadherin interactions. The level of adhesion can then be quantified with and without a β-cadherin antibody. Adhesion as a function of antibody concentration is then used to determine an IC50 value. Example 3 provides further details of a β-cadherin-dependent cell adhesion assay that was used to measure IC50 values for β-cadherin antibodies.

Inhibition of P-cadherin activity can also be measured using a P-cadherin-dependent spheroid disruption assay. This type of assay measures the ability of a P-cadherin antibody to disrupt preformed P-cadherin-dependent cellular aggregations. An IC50 value can be determined by measuring the size reduction of aggregates as a function of antibody concentration. Example 5 provides further details of a β-cadherin-dependent spheroid disruption assay that was used to measure IC50 values for β-cadherin antibodies. The methods and assays described above for determining the inhibition of β-cadherin activity by different antibodies or antigen-binding portions thereof can be used during both the first screening stage and during subsequent optimization stages.

5 Molecular selectivity

The selectivity of the β-cadherin antibodies of the present invention over other cadherins, such as E-cadherin, can be determined using methods well known in the art. The selectivity can be determined using, for example, Western blot, flow cytometry, ELISA, immunoprecipitation or RIA. Example 7 provides further details of an ELISA assay that was used to measure the selectivity of specific antibodies to P-15 cadherin over E-cadherin. The methods and assays described above for determining the selectivity to β-cadherin of different antibodies or anti-binding portions thereof can be used during both the first screening stage and during subsequent optimizing stages.

Pharmaceutical preparations and administration

This invention also relates to a pharmaceutical composition for the treatment of abnormal cell growth in a mammal, including a human, comprising an amount of a β-cadherin antibody or antigen-binding portion thereof, as described herein, which is effective in treating abnormal cell growth, and a pharmaceutically acceptable carrier.

The antibodies and antigen-binding portions of the present invention can be incorporated into pharmaceutical compositions suitable for administration to a patient. The pharmaceutical composition typically comprises an antibody or antigen-binding portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media / dispersion media, coating (s), antibacterial agents / anti-bacterial agents, and antifungal agents / antifungal agents, isotope (s) n (e) and absorption-delaying agent (s) and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, physiological saline, phosphate buffered physiological saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or relatively small amounts of auxiliaries such as wetting agents or emulsifiers, preservatives or buffers, which increase the shelf life or the effectiveness of the antibody.

The compositions of this invention can take a variety of forms, 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 depends on the intended method of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred route of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure that is suitable for a high drug concentration. Sterile injectable solutions can be prepared by incorporating the β-cadherin antibody in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Usually, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods for preparation are vacuum drying and freeze drying, which yields a powder of the active ingredient plus any additional desired ingredient that comes 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 a dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by incorporating into the composition an agent that delays absorption, for example monostearate salts and gelatin.

80

The antibodies or antibody portions of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is subcutaneous, intramuscular or intravenous infusion. . As will be recognized by those skilled in the art, the route and / or method of administration will vary depending on the desired results.

In certain embodiments, the antibody compositions of the present invention can be prepared with a carrier that will protect the antibody against rapid release, such as a release-regulated preparation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many processes for the preparation of such compositions are usually known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, which is incorporated herein by reference.

Additional active compounds can also be incorporated into the compositions. In certain embodiments, an inhibitory β-cadherin antibody of the invention is co-formulated with, and / or co-administered with, one or more additional therapeutic agents. These agents include, without limitation, antibodies that bind other targets, anti-tumor agents, anti-angiogenesis agents, signal transduction inhibitors, anti-proliferative agents, chemotherapeutic agents or peptide analogs that inhibit β-cadherin. Such combination therapies may require lower doses of both the inhibitory P-35 cadherin antibody and co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapy.

The compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactic effective amount" of an antibody or antigen-binding portion of the invention. A "therapeutically effective amount" refers to an amount that, at dosages and for time periods that are necessary, is effective to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antigen-binding portion can vary according to factors such as the disease state, age, gender and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the antibody or antigen binding portion are offset by the therapeutically beneficial effects. A "prophylactic effective amount" refers to an amount that, at dosages and for time periods that are necessary, is effective to achieve the desired prophylactic result. Since a prophylactic dose is used in patients prior to or at an earlier stage of the disease, the prophylactically effective amount may typically be less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be delivered over time, or the dose can be reduced or increased proportionally, as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

ring. Dosage unit form, as used herein, refers to physically discrete units suitable as unit doses for the mammalian subjects to be treated; wherein each unit contains a predetermined amount of active compound that is calculated to produce, together with the required pharmaceutical carrier, the desired therapeutic effect. The specification for the dosage unit forms of the invention is dictated by, and directly depends on, (a) the unique characteristics of the β-cadherin antibody or portion thereof and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technique of preparing such an antibody for the treatment of sensitivity in individuals.

15 A distinctive, non-restrictive trajectory for a

therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.025 to 50 mg / kg, more preferably 0.1 to 50 mg / kg, more preferably 0.1-25, 0.1 to 10 or 0, 1 to 3 mg / kg. In some embodiments, a composition contains 5 mg / ml of antibody in a buffer of 20 mM sodium citrate, pH

5.5, 140 mM NaCl and 0.2 mg / ml polysorbate 80. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated. It should further be understood that for any specific patient, specific dosage regimens should be adjusted over time in accordance with the individual needs and professional judgment of the person administering the preparations or supervising the administration of the compositions, and that dosage ranges set forth herein are merely characterizing and are not intended to limit the scope or practice of the composition for which protection is sought.

Another aspect of the present invention provides kits that include a β-cadherin antibody or antigen-binding portion of the invention, or a composition comprising such an antibody or portion. A kit may include, in addition to the antibody or preparation, diagnostic or therapeutic agents. A kit may also include instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit comprises the antibody or a composition comprising it, and a diagnostic agent that can be used in a method described below. In another preferred embodiment, the kit comprises the antibody or composition comprising it, and one or more therapeutic agents that can be used in a method described below.

Diagnostic methods for use

The β-cadherin antibodies or antigen-binding portions thereof can be used in diagnostic methods for detecting β-cadherin in vitro or in vivo in a biological sample. For example, the β-cadherin antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot or immunoprecipitation. The β-cadherin antibodies of the invention can be used to detect β-cadherin derived from humans. The β-cadherin antibodies can also be used to detect β-cadherin derived from mice, rats, and cynomolgus monkeys.

The invention provides a method for detecting β-cadherin in a biological sample, comprising contacting the biological sample with a β-cadherin antibody of the invention and detecting the bound antibody. In one embodiment, the β-cadherin antibody is directly labeled with a detectable label. In another embodiment, the β-cadherin antibody (the first antibody) is unlabelled and a second antibody or other molecule capable of binding the β-cadherin antibody is labeled. As is well known to those skilled in the art, a second 84 antibody is selected which is capable of specifically binding the specific type and class of the first antibody. For example, if the β-cadherin antibody is a human IgG, then the secondary antibody could be an anti-5i-human IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are commercially available, e.g. from Pierce Chemical Co.

Suitable labels for the antibody or secondary antibody have been previously discussed and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholine esterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescent cell, fluorescein isothiocyanate, rhodamine, dichloro-triazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material comprising luminol; and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.

In other embodiments, P-cadherin can be tested in a biological sample by competitive immunoassay using P-cadherin standards labeled with a detectable substance and an unlabelled P-cadherin antibody. In this assay, the biological sample, the labeled P-cadherin standards and the P-cadherin antibody are combined, and the amount of labeled P-cadherin standard bound to the unlabelled antibody is determined. The amount of β-cadherin in the biological sample is inversely proportional to the amount of labeled β-cadherin standard bound to the β-cadherin antibody.

85

The immunoassays described above can be used for a number of purposes. For example, the β-cadherin antibodies can be used to detect β-cadherin in cultured cells. In a preferred embodiment, the β-cadherin antibodies are used to determine the amount of β-cadherin produced by cells treated with different compounds. This method can be used to identify compounds that modulate β-cadherin protein levels. According to this method, one sample of cells is treated with a test compound for a period of time, while another sample is left untreated. If the total level of β-cadherin is to be measured, the cells are lysed and the total β-cadherin level is measured using one of the immunoassays described above. The total level of β-cadherin in the treated versus the untreated cells is compared to determine the effect of the test compound.

A preferred immunoassay for measuring total P-cadherin levels is flow cytometry or immunohistochemistry. Methods such as ELISA, RIA, flow cytometry, Western blot, immunohistochemistry, cell surface labeling of integral membrane proteins and immunoprecipitation are well known in the art. See, e.g., Harlow and Lane, supra. In addition, the immunoassays can be increased for high throughput screening to test a large number of compounds for either activation or inhibition of β-cadherin expression.

The β-cadherin antibodies of the invention can also be used to determine the levels of β-cadherin in a tissue or in cells derived from the tissue. In some embodiments, the tissue is a diseased tissue. In some embodiments of the method, a tissue or a biopsy thereof is excised from a patient. The tissue or biopsy is then used in an immunoassay to determine, for example, total β-cadherin levels or the localization of β-cadherin using the methods discussed above.

The antibodies of the present invention can also be used in vivo to identify tissues and organs expressing β-cadherin. One advantage of using the human β-cadherin antibodies of the present invention is that they can be used safely in vivo, without eliciting a significant immune response to the antibody upon administration, other than antibodies of non-human origin or with humanized or chimeric antibodies.

The method comprises the steps of administering a P-cadherin antibody that is detectably labeled or a composition comprising it, to a patient in need of such a diagnostic test, and subjecting the patient to imaging analysis to determine the Site of the β-cadherin-expressing tissues. Imaging analysis is well known in the medical art and includes, without limitation, X-ray analysis, magnetic resonance imaging (MRI) or computed tomography (CT). The antibody can be labeled with any agent suitable for in vivo imaging, for example, a contrast agent such as barium that can be used for X-ray analysis, or a magnetic contrast agent such as a gadolinium chelate that can be used for MRI or CT . Other labeling agents include, without limitation, radioisotopes such as "Tc. In another embodiment, the β-cadherin antibody will be unlabelled and will be eliminated by administering a second antibody or other molecule that is detectable and that the β-cadherin antibody can bind In one embodiment, a biopsy is obtained from the patient to determine whether the tissue of interest expresses β-cadherin.

87

Therapeutic methods of use

In another embodiment, the invention provides a method for inhibiting β-cadherin activity by administering a β-cadherin antibody to a patient in need thereof. Any of the antibodies or antigen-binding portions thereof described herein may be used therapeutically. In a preferred embodiment, the β-cadherin antibody is a human, chimeric or humanized antibody. In another preferred embodiment, the β-cadherin is human and the patient is a human patient. Alternatively, the patient may be a mammal expressing a P-cadhrin with which the P-cadherin antibody cross-reacts. The antibody can be administered to a non-human mammal expressing β-cadherin with which the antibody cross-reacts (e.g., a rat, a mouse or a cynomolgus monkey), for veterinary purposes or as an animal model for human disease. Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of this invention.

In another embodiment, a β-cadherin antibody or antibody portion thereof can be administered to a patient expressing inappropriate high levels of β-cadherin. The antibody can be administered once, but is more preferably administered multiple times. The antibody can be administered from three times a day to once every six months or more. Administration can be in a schedule such as three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month , once every two months, once every three months and once every six months. The antibody can also be administered continuously via a mini pump. The antibody can be administered via a mucosa, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral or intratumor route. The antibody can be administered one 88 times, at least twice, or for at least the time period until the condition is treated, alleviated or cured. The antibody will usually be administered as long as the condition is present. The antibody will usually be administered as a part of a pharmaceutical preparation, such as aupra described. The dosage of the antibody will usually be in the range of 0.1 to 100 mg / kg, more preferably 0.5 to 50 mg / kg, more preferably 1 to 20 mg / kg and even more preferably 1 to 10 mg / kg. The serum concentration of the antibody can be measured by any method known in the art.

This invention also relates to a method for treating abnormal cell growth in a mammal, including a human, comprising administering to that mammal a therapeutically effective amount of a β-cadherin antibody or antigen-binding portion thereof, as described herein, that is effective is in treating abnormal cell growth.

In one embodiment of this method, abnormal cell growth is cancer, including, but not limited to, mesothelioma, hepatobiliary (liver and bile) tumor, a primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, skin or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal area, stomach cancer, gastrointestinal (gastric, colorectal) and duo-30 denal) cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the thin intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testes cancer, 89 chronic or a cute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system neoplasms (CNS), primary CNS lymphoma, non-Hodgkin lymphoma, spinal tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination of one or more of the preceding cancers.

In a preferred embodiment of the present invention, the cancer is selected from lung cancer (NSCLC and SCLC), head or neck cancer, ovarian cancer, colon cancer, rectal cancer, cancer of the anal area, stomach cancer, breast cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, central nervous system neo-plasmas, primary CNS lymphoma, non-Hodgkin's lymphoma, spinal cancer, or a combination of one or more of the foregoing may-2 0 cherry.

In another preferred embodiment of the present invention, the cancer is selected from lung cancer (NSCLC and SCLC), ovarian cancer, colon cancer, rectal cancer, cancer of the anal area, or a combination of one or more of the foregoing cancers.

In another embodiment of that method, that abnormal cell growth is a benign proliferative disease, including, but not limited to, psoriasis, benign prostatic hypertrophy or restinosis.

This invention also relates to a method for treating abnormal cell growth in a mammal, administering to that mammal an amount of a β-cadherin antibody or antigen-binding portion thereof, as described herein, that is effective in treating abnormal cell growth in combination with an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents substances, anti-hormones and anti-androgenic hormones, included.

The invention also relates to a pharmaceutical composition for the treatment of abnormal cell growth in a mammal, including a human, comprising an amount of a β-cadherin antibody or antigen-binding portion thereof, as described herein, which is effective in treating abnormal cell growth in combination with a pharmaceutically acceptable carrier and an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones and anti-androgen hormones.

The invention also relates to a method for the treatment of a hyperproliferative disorder in a mammal which administers to that mammal a therapeutically effective amount of a β-cadherin antibody or antigen-binding portion thereof, as described herein, in combination with an anti-tumor -25 gens selected from the group consisting of anti-proliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-I inhibitors, cox-II inhibitors, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, radiation, 30 cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents, anti-hormones, statins, and anti-androgen hormones.

In one embodiment of the present invention, the anti-tumor agent used in combination with a β-cadherin antibody or antigen-binding portion thereof, and pharmaceutical compositions described herein, is an anti-angiogenesis agent, kinase inhibitor, pan-kinase inhibitor or growth factor inhibitor. Preferred pan-kinase inhibitors include SU-11248 described in U.S. Patent 6,573,293 (Pfizer, Inc., NY, USA).

Antibiogenesis agents include, but are not limited to, the following agents, such as EGF inhibitor, EGFR inhibitors, VEGF inhibitors, VEGFR inhibitors, TIE2 inhibitors, IGF1R inhibitors, COX-II (cyclooxygenase II) inhibitors , MMP-2 (matrix-metalloproteinase 2) inhibitors and MMP-9 (matrix-10 metalloproteinase 9) inhibitors. Preferred VEGF inhibitors include, for example, Avastin (bevacizumab), an anti-VEGF monoclonal antibody from Genentech, Inc. from South San Francisco, California.

Additional VEGF inhibitors include CP-547,632 (Pfizer Inc., NY, USA), AG13736 (Pfizer Inc.), ZD-6474 (AstraZeneca), AEE788 (Novartis), AZD-2171), VEGF Trap (Regeneron, / Aventis), Vatalanib (also known as PTK-787, ZK-222584: Novartis & Schering AG), Macugen (pegaptanib-octa sodium, NX-1838, EYE-001, Pfizer Ine./Gilead/Eyetech), IM862 (Cytran Inc. of Kirkland, Washington, USA) and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California) and combinations thereof. VEGF inhibitors useful in the practice of the present invention are described in U.S. Patent Nos. 6,534,524 and 6,235,764, both of which are incorporated in their entirety for all purposes. Particularly preferred VEGF inhibitors include CP-547.632, AG13736, Vatalanib, Macugen and combinations thereof.

Additional VEGF inhibitors are described in, for example, WO 99/24440 (published May 20, 1999), international PCT application PCT / IB99 / 00797 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995 ), WO 99/61422 (published December 2, 1999), U.S. Patent 6,534,524 (describes AG13736), U.S. Patent 5,834,504 (issued November 10, 1998), WO 98/50356 (published November 12, 1998), U.S. Patent No. r \

Zf £ *

5,883,113 (issued March 16, 1999), U.S. Patent 5,886,020 (released March 23, 1999), U.S. Patent 5,792,783 (issued August 11, 1998), US Patent 6,653,308 (released November 25, 2003), WO 99/10349 (published on March 4, 1999), WO

97/32856 (published on September 12, 1997), WO 97/22596 (published on June 26, 1997), WO 98/54093 (published on December 3, 1998), WO 98/02438 (published on January 22, 1998), WO 99 / 16755 (published on April 8, 1999) and WO 98/02437 (published on January 22, 1998), all of which are incorporated herein by reference in their entirety.

Other antiproliferative agents that can be used with the antibodies, or antigen-binding portions thereof, of the present invention include inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFr, including the compounds described, and for which protection is sought, in the following U.S. patent applications: 09/221946 (filed December 28, 1998); 09/454058 (served in 20 on December 2, 1999); 09/501163 (filed February 9, 2000); 09/539930 (filed on March 31, 2000); 09/202796 (filed May 22, 1997); 09/384339 (filed August 26, 1999) and 09/383755 (filed August 26, 1999); and the compounds described, and for which protection is sought, in the following U.S. provisional patent applications: 60/168207 (filed November 30, 1999); 60/170119 (filed December 10, 1999); 60/177718 (filed January 21, 2000); 60/168217 (filed November 30, 1999) and 60/200834 (filed May 30, 2000). Each of the preceding patent applications and provisional patent applications is incorporated herein by reference in its entirety.

PDGFr inhibitors include, but are not limited to, those described in WO01 / 40217, published July 7, 2001 and WO2004 / 020431, published on March 11, 2004, the contents of which are incorporated in their entirety for all purposes. Preferred PDGFr inhibitors include 93

Pfizer's CP-673.451 and CP-868.596, and are pharmaceutically acceptable salts.

Preferred GARF inhibitors include Pfizer's AG-2037 (pe-litrexol and its pharmaceutically acceptable salts). GARF-5 inhibitors useful in the practice of the present invention are described in U.S. Patent 5,608,082, which is incorporated in its entirety for all purposes.

Examples of useful COX-II inhibitors that can be used in combination with a β-cadherin antibody or antigen-binding portion thereof, as described herein, and pharmaceutical compositions described herein include CELEBREX ™ (celecoxib), parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189 (Lumiracoxib), BMS 347070, RS 57067, NS-398, Bextra (val-decoxib), paracoxib, Vioxx (rofecoxib), SD-8381, 4 -methyl-2- (3,4-dimethylphenyl) -1- (4-sulfamoylphenyl) -1H-pyrrole, 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole, T -614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125 and Arcoxia (etoricoxib). Furthermore, COX-II inhibitors are described in U.S. Patent Applications 10 / 801,446 and 10 / 801,429, the contents of which are incorporated in their entirety for all purposes.

In one preferred embodiment, the anti-tumor agent is celecoxib, as described in U.S. Patent 5,466,823, the contents of which are incorporated by reference in their entirety for all purposes. The structure for Celecoxib is shown below:%> ibsCv-> \ - = J celecoxib / CAS No. 169590-42-5 5,466,623 C-2779 SC-5B635 h3c 35 In one preferred embodiment, the anti-tumor agent is valdecoxib, as described in U.S. Patent 5,633,272, the contents of which are incorporated in their entirety in reference 94 for all purposes. The structure for valdecoxib is shown below: "/" * N valdecoxib / \ CAS No. 161695 * 72-7 f \ 5,633,272 W / C-2665 SC-65672

In one preferred embodiment, the anti-tumor agent 10 is parecoxib, as described in U.S. Patent 5,932,598, the contents of which are incorporated by reference in their entirety for all purposes. The structure for parecoxib is shown below: r / Γ \ parecoxib \ 9 CAS no. 198470-84-7

In one preferred embodiment, the anti-tumor agent is deracoxib, as described in U.S. Patent 5,521,207, the contents of which are incorporated by reference in their entirety for all purposes. The structure for deracoxib is shown below: deracoxib F / CAS No. 169590-41-4 ^ ✓ 5,521,207

C 2779 H 3 C-O

30

In one preferred embodiment, the anti-tumor agent is SD-8381, as described in U.S. Patent 6,034,256, the contents of which are incorporated by reference in their entirety for all purposes. The structure for SD-8381 I3 SD-8381 C1 6.034.256

Ex. 175 95

In one preferred embodiment, the anti-tumor agent is ABT-963, as described in international publication WO 2002/24719, the contents of which are incorporated by reference in their entirety for all purposes. The structure for ABT-963 is shown below: ABT-963 I WO 00/24719

In one preferred embodiment, the anti-tumor agent is rofecoxib, as shown below: 162011-90-7 20

In one preferred embodiment, the anti-tumor agent is MK-663 (etoricoxib), as described in international publication WO 1998/03484, the contents of which are incorporated by reference in their entirety for all purposes. The structure for etoricoxib is shown below: ° V °

rrS

MK-663 L II etoricoxib CAS No. 202409-33-4 WO 98/03484 n * tch3 ^ 86218 30

In one preferred embodiment, the anti-tumor agent is COX-189 (Lumiracoxib), as described in international publication WO 1999/11605, the contents of which are incorporated by reference in their entirety for all purposes. The structure for Lumiracoxib is shown below: co2h "ό: C * I <*» 1 - COX-189 ^

Lumiracoxlb CAS No. 220991-20-8 Novartis NO 99/11605 In one preferred embodiment, the anti-tumor agent is BMS-347070, as described in U.S. Patent 6,180,651, the contents of which are incorporated by reference in their entirety for all purposes. The structure for BMS-347070 is shown below: SOjCH, BMS 347070 IC CAS No. 10. 197438-48-5 -13 6,180,651

In one preferred embodiment, the anti-tumor agent is NS-398 (CAS 123653-11-2). The structure for NS-398 (CAS 123653-11-2) is shown below: HN-sC NS-398.

CAS No. 123653-11-2 In one preferred embodiment, the anti-tumor agent is RS 57067 (CAS 17932-91-3). The structure for RS-57067 (CAS 17932-91-3) is shown below: RS 57067 CAS no. 17932-91-3

In one preferred embodiment, the anti-tumor agent is 4-methyl-2- (3,4-dimethylphenyl) -1- (4-sulfamoylphenyl) -1 H-pyrrole. The structure for 4-methyl-2- (3,4-dimethylphenyl) -1- (4-sulfamoylphenyl) -pyrrole is shown below: ch3 SO3 NH,

In one preferred embodiment, the anti-tumor agent is 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1 H-pyrrole.

The structure for 2 - (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole is shown below: ch3, c10 o -N ^ Λ, SO2 OH2

In one preferred embodiment, the anti-tumor agent is meloxicam. The structure for meloxicam is shown below: rrSV ^

Meloxicam Other useful inhibitors such as anti-tumor agents, used in combination with antibodies of the present invention and pharmaceutical compositions described herein include aspirin, and non-steroidal anti-inflammatory drugs (non-steroidal anti-inflammatory drugs) (NSAIDs) that inhibit the enzyme that makes prostaglandins (cyclooxygenase I and II), leading to lower levels of prostaglandins, include, but are not limited to, the following, Salsalate (Amigesic), Diflunisal (Dolobid), Ibuprofen (Motrin), Ketoprofen (Orudis), Nabu-30 metone (Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn), Diclofenac (Voltaren), Indomethacin (Indocin), Sulindac (Clinoril), Tolmetin (Tolectin), Etodolac (Lodi-ne) , Ketorolac (Toradol), Oxaprozin (Daypro) and combinations thereof. Preferred COX-I inhibitors include ibuprofen (Motrin), nuprin, naproxen (Aleve), indomethacin (Indocin), nabumeton (Relafen) and combinations thereof.

98

Targeted agents used in combination with a β-cadherin antibody or antigen-binding portion thereof, as described herein, and pharmaceutical compositions thereof, as described herein, include EGFr inhibitors such as Iressa (gefitinib, AstraZeneca), Tarceva (erlotinib or OSI-774, OSI Pharmaceuticals Inc.) / Erbitux (cetuximab, Imclone Pharmaceuticals, Inc.), EMD-7200 (Merck AG), ABX-EGF (Amgen Inc. and Abgenix Inc.), HR3 (Cu-10 lane government ), IgA antibodies (University of Erlangen-Nuremberg), TP-38 (IVAX), EGFR fusion protein, EGF vaccine, anti-EGFr immunoliposomes (Hermes Biosciences Inc.) and combinations thereof.

Preferred EGFr inhibitors include Iressa, Erbitux, Tar-15 ceva and combinations thereof.

The present invention also relates to anti-tumor agents selected from pan-erb receptor inhibitors or ErbB2 receptor inhibitors, such as CP-724.714 (Pfizer, Inc.), CI-1033 (canertinib, Pfizer, Ine.), Herceptin 20 (trastuzumab, Genentech Inc.), Omitarg (2C4, pertuzumab, Genentech Inc.), TAK-165 (Takeda), GW-572016 (lonafarnib,

GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth), PKI-166 (Novartis), dHER2 (HER2 Vaccine, Corixa and GlaxoSmithKline), APC8024 (HER2 Vaccine, Dendreon), an-25 ti-HER2 -bispecific antibody (Decof Cancer Center), B7.her2.IgG3 (Agensys), AS HER2 (Research Institute for Rad Biology & Medicine), trifunctional bispecific antibodies (University of Munich) and mAB AR-209 (Aronex Pharmaceuticals Inc.). ) and mAB 2B-1 (Chiron) and combinations thereof. Preferred erb-selective anti-tumor agents include Herceptin, TAK-165, CP-724,714, ABX-EGF, HER3 and combinations thereof. Preferred pan-erb receptor inhibitors include GW572016, CI-1033, EKB-569, and

Omitarg and combinations thereof.

Additional erbB2 inhibitors include those that become

described in WO 98/02434 (published on January 22, 1998), WO 99/35146 (published on July 15, 1999), WO

99 99/35132 (published on July 15, 1999), WO 98/02437 (published on January 22, 1998), WO 97/13760 (published on April 17, 1997), WO 95/19970 (published on July 27, 1995), U.S. Patent 5,587,458 (issued on December 24, 1996) and U.S. Patent 5,877,305 (released on March 2, 1999), each of which is hereby incorporated by reference in its entirety. ErbB2 receptor inhibitors useful in the present invention are also described in U.S. Patents 6,465,449 and 6,284,764, and International Application WO 2001/98277, each of which is incorporated herein by reference in its entirety.

Furthermore, other anti-tumor agents can be selected from the following agents, BAY-43-9006 (Onyx Pharmaceuticals 15 Inc.), Genasense (augmeroses, Genta), Panitumumab (Abgenix / Amgen), Zevalin (Schering), Bexxar (Cerixa) / GlaxoSmithKline), Abarelix, Alimta, EPO 906 (Novartis), discodermolide (XAA-296), ABT-510 (Abbott), Neopastat (Aeterna), enzastaurine (Eli Lilly), Combrestatin 20 A4P (Oxigene), ZD- 6126 (AstraZeneca), flavopiridol (Avenis), CYC-202 (Cyclacel), AVE-8062 (Aventis), DMXAA (Roche / Antisoma), Thymitaq (Eximias), Temodar (temozolomide, Schering Plow) and Revilimd ( Celegene) and combinations thereof.

Other anti-tumor agents may be selected from the following agents, CyPat (cyproterone acetate), Histerelin (histrelin acetate), Plenaixis (abarelix depot), Atrasentan (ABT-627), Satraplatin (JM-216), thalomide (Thalidomide), Theratope DP, Temilene DP ), ABI-007 (paclitaxel), Evista (raloxifene), Atamestane (Biomed-777), Xyotax (polygluta-size paclitaxel), Targetin (bexarotine) and combinations thereof.

Furthermore, other anti-tumor agents may be selected from the following agents, Trizaone (tirapazamine), Aposyn 35 (exisulind), Nevastat (AE-941), Ceplene (histamine dihydrogen chloride), Orathecin (rubitecan), Virulizin, Gastrimmune (G17DT), DX-8951f (exatecan methanesulfonate), Onconase 100 (ranpirnase), BEC2 (mitumoab), Xcytrin (motexafingadolinium) and combinations thereof.

Further anti-tumor agents can be selected from the following agents, CeaVac (CEA), NeuTrexin (trimetresate-glucuronate) and combinations thereof. Additional anti-tumor agents can be selected from the following agents, OvaRex (oregovomab), Osidem (IDM-1) and combinations thereof. Additional anti-tumor agents can be selected from the following agents, Advexin (ING 201), Tirazone (tirapa-zamine) and combinations thereof. Additional anti-tumor agents can be selected from the following agents, RSR13 (efaproxiral), Cotara (1311 chTNT 1 / b), NBI-3001 (IL-4) and combinations thereof. Additional anti-tumor agents can be selected from the following agents, Canvaxin, GMK-15 vaccine, PEG Interon A, Taxoprexin (DHA / paclitaxel) and combinations thereof. Other preferred anti-tumor agents include Pfizer's MEK1 / 2 inhibitor PD325901, Array Biopharm's MEK inhibitor ARRY-142886, Bristol Myers' CDK2 inhibitor BMS-387,032, Pfizer's CDK inhibitor PD0332991 and AstraZeneca's AXD-5438 and combinations thereof. Furthermore, use may also be made of mTOR inhibitors such as CCI-779 (Wyeth) and rapamycin derivatives RAD001 (Novartis) and AP-23573 (Ariad), HDAC inhibitors SAHA (Merck Inc./Aton Pharmaceuticals) and combinations thereof. Additional anti-tumor agents include the aurora 2 inhibitor VX-680 (Vertex), Chkl / 2 inhibitor XL844 (Exilixis).

The following cytotoxic agents, e.g., one or more selected from the group consisting of epirubicin (El-lence), docetaxel (Taxotere), paclitaxel, Zinecard (dexra-30 zoxan), rituximab (Rituxan), imatinibmethane sulfonate (Gleevec) and combinations thereof, can be used in combination with a β-cadherin antibody or antigen-binding portion thereof, as described herein, and pharmaceutical compositions thereof, as described herein.

The invention also contemplates the use of the antibodies and antigen-binding portions thereof of the present invention, together with hormonal therapy, including, but not limited to, exemestan (Aromasin, Pfizer Ine.), Leuproreline (Lupron or Leuplin, TAP / Abbott / Takeda), anastrozole (Arimidex, Astrazeneca), gosrelin (Zoladex, AstraZeneca), doxercalciferol, fadro-5 zol, formestan, tamoxifen citrate (tamoxifen, Nolvadex,

AstraZeneca), Casodex (AstraZeneca), Abarelix (Praecis), Trelstar, and combinations thereof.

The invention also relates to agents of hormonal therapy such as antiestrogens, including, but not limited to, fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole (Femara, Novartis), anti-androgen hormones such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex® (4 '- cyano-3 - (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-31- (trifluoromethyl) propionanilide, bica-lutamide) and combinations thereof.

Furthermore, the invention provides antibodies of the present invention alone or in combination with one or more supportive care products, eg a product selected from the group consisting of Filgrastim (Neupogen), ondansetron (Zofran), Fragmin, Procrit, Aloxi , Emend, or combinations thereof.

Particularly preferred cytotoxic agents include Camptosar, Erbitux, Iressa, Gleevec, Taxotere, and combinations thereof.

The following topoisomerase I inhibitors can be used as anti-tumor agents, camptothecin, irinotecan HCl (Camptosar), edotecarin, orathecine (Su-pergen), exatecan (Daiichi), BN-80915 (Roche) and combinations thereof. Particularly preferred topoisomerase II inhibitors include epirubicin (Ellence).

The antibodies of the invention can be used with anti-tumor agents, alkylating agents, anti-tabolites, antibiotics, plant-derived anti-tumor agents, camptothecin derivatives, tyrosine kinase inhibitors, other antibodies, interferons, and / or biological response modifiers.

102

Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan, mitobronitol, carboquon, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, altretamine, AP-5280, apaziquine, brittle , bendamus tine, carmustine, estramustine, photemustine, glufosfamide, ifosfamide, KW-2170, mafosfamide, and mitolactol; platinum-coordinated alkylating compounds include, but are not limited to, cisplatin, Paraplatin (carboplatin), eptaplatin, lobaplatin, nedaplatin, Eloxatin (oxaliplatin, Sanofi) or satrplatin, and combinations thereof. Particularly preferred alkylating agents include Eloxatin (oxaliplatin).

Antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil (5-FU) alone or in combination with leucovorine, tegafur, UFT, doxifluridine, carmofur, cyta-rabine, cytarabineocphosphate , enocitabine, S1, Alimta (premetrexed disodium, LY231514, MTA), Gemzar (gemcitabi-nein, Eli Lilly), fludarabine, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethyto-nyxy-numine-nyl-hydroxyide, nyxy-cytosine n-hydroxy , TS-1, melphalan, nelarabine, nolatrexed, phosphate, disodium-premetrexed, pentostatin, pelitrexol, raltitrexed, tria-pine, trimetrexate, vidarabine, vincristine, vinorelbine; or for example one of the preferred antimetabolites described in European patent application 239362, such as N- (5- [N- (3,4-dihydro-2-methyl-4-oxo-quinazolin-6-ylmethyl) -N-methylamino] -2- thenoyl) -L-glutamic acid and combinations thereof.

Antibiotics include intercalating antibiotics, but are not limited to: aclarubicin, actinomycin D, amberbicin, annamycin, adriamycin, bleomycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, galarubicin, neocarbicin, mitocycin, mitocycin, mitocycin statin, peplomycin, pirarubicin, rebeccamycin, stimulant, streptozocin, valrubicin, zinostatin and combinations thereof.

Plant-derived anti-tumor substances include, for example, those selected from mitotic inhibitors, e.g., vinblastine, docetaxel (Taxotere), paclitaxel, and combinations thereof.

Cytotoxic topoisomerase inhibiting agents include one or more agents selected from the group consisting of aclarubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomo-tecan, irinotecan HCl (Camptosar), edirubicarine ne (Ellence), etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirarubicin, pixantrone, rubitecan, sobu-zoxan, SN-38, tafluposide, topotecan, and combinations thereof.

Preferred cytotoxic topoisomerase inhibiting agents include one or more agents selected from the group consisting of camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan HCl (Camptosar), edotecarin, epirubicin (Ellence), etoposide, SN-38, topotecan, and combinations thereof.

Immunological substances include interferons and numerous other immune enhancing agents. Interferons include interferon alpha, interferon alpha-2a, interferon a1-fa-2b, interferon beta, interferon gamma-la, interferon gamma-1b (Actimmune) or interferon gamma-nl and combinations thereof. Other agents include filgrastim, lentinan, si-zofilan, TheraCys, ubenimex, WF-10, aldesleukin, alemtu-zumab, BAM-002, dacarbazine, daclizumab, denileukin, gem-tuzumab, ozogamicin, ibritumomab, imiquimod lentog, imiquimod lentog melanoma vaccine (Corixa), molgramostim, OncoVAX-CL, sargramostim, tasonermin, tecleukin, thymalasin, to-situmomab, Virulizin, Z-100, epratuzumab, mitumomab, ore-govomab, pemtumomab (Y-muHonGD) (D-muHonGD) (D-muHonGG) (D-muHon) combinations thereof.

Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses, such as survival, growth, or differentiation of tissue cells, thereby leading them to have anti-tumor activity. Such agents include krestin, lentinan, sizofiran, picibanil, ubenimex and combinations thereof.

Other anticancer agents include alitretinoin, ampligene, atrasentan, bexarotene, bortezomib, bosentan, calcitriol, exisulind, finasteride, photemustine, ibandanoic acid, spleen phosphine, mitoxantrone, 1-asparaginase, pro-10 carbazine, dacarbazine, pentaegarbase, pentecargamide, pentazinarbase Telcyta (TLK-286, Telik Ine.), Velcade (bortemazib, Millenium), tretinoin and combinations thereof.

Other antiangiogenic compounds include acitretin, fenretinide, thalidomide, zoledronic acid, angiostatin, aplidine, cilengtide, combretastatin A-4, endostatin, halofuginone, rebimastat, removab, Revlimid, squalamine, ukrain, Vitaxin and combinations thereof .

Platinum-coordinated compounds include, but are not limited to, cisplatin, carboplatin, nedaplastin, oxaliplatin, and combinations thereof.

Camptothecin derivatives include, but are not limited to, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan, and combinations thereof.

Other anti-tumor agents include mitoxantrone, 1-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pentostatin, tretinoin, and combinations thereof.

Use can also be made of anti-tumor agents capable of enhancing anti-tumor immune responses, such as CTLA4 (cytotoxic lymphocyte antigen 4) antibodies, and other agents capable of blocking CTLA4, such as MDX-010 ( Medarex) and CTLA4 compounds described in U.S. Patent 6,682,736; and anti-proliferative agents such as other farnesyl protein transferase inhibitors, for example the farnesyl protein transferase inhibitors. Additional, specific CTLA4-105 antibodies that can be used in the present invention include those described in U.S. Provisional Application 60 / 113,647 (filed December 23, 1998), U.S. Patent 6,682,736, both of which are herein incorporated by reference in their entirety. included .

Specific IGFIR antibodies that can be used in the present invention include those described in international patent application WO 10 2002/053596, which is incorporated herein by reference in its entirety.

Specific CD40 antibodies that can be used in the present invention include those described in international patent application WO 2003/040170, which is incorporated herein by reference in its entirety.

Gene therapy agents can also be used as anti-tumor agents, such as TNFerade (GeneVec), which express TNF-alpha in response to radiotherapy.

In one embodiment of the present invention, statins can be used in combination with a β-cadherin antibody or antigen-binding portion thereof, as described herein, and pharmaceutical compositions thereof. Statins (HMG-CoA reductase inhibitors) can be selected from the group consisting of Atorvastatin (Lipitor, Pfizer Ine.), Provastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Ine.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin (Crestor, AstraZe-neca), Lovostatin and Niacin (Advicor, Kos Pharmaceuticals), derivatives and combinations thereof.

In a preferred embodiment, the statin is selected from the group consisting of Atovorstatin and Lovastatin, derivatives and combinations thereof.

Other agents useful as anti-tumor agents include Caduet.

106

For any of the methods for treating a hyperproliferative disorder or abnormal cell growth as described herein, using a combination of a P-cadherin antibody or antigen-binding portion with at least one additional therapeutic agent, the P-cadherin antibody may be conjugated, or derivatized, with the additional therapeutic agent. The at least one additional therapeutic agent can also be administered separately, or in a non-derivatized or non-conjugated manner. When the at least one additional therapeutic agent is not derivatized or conjugated to the antibody, it can be administered within the same pharmaceutical composition as the antibody, or it can be administered in a separate composition. Gene therapy

The nucleic acid molecules encoding the antibodies and antibody portions of the present invention can be administered via gene therapy to a patient who needs them. The therapy can be done either in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding both a heavy chain and a light chain are administered to a patient. In a more preferred embodiment, the nucleic acid molecules are administered such that they are stably integrated into chromosomes of B cells, because these cells are specialized for producing antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and re-transplanted into a patient in need thereof. In another embodiment, precursor B cells or other cells are infected in vivo using a virus known to infect the cell type of interest. Typical vectors used for gene therapy include liposomes, plasmids, and viral vectors. Typical viral vectors are retroviruses, adenoviruses and adeno-associated 107 viruses. After either in vivo or ex vivo infection, levels of antibody expression can be monitored by taking a sample from the treated patient and using any immunoassay known in the art or discussed herein.

In a preferred embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain or an antigen-binding portion thereof of a β-cadherin antibody, and expressing the nucleic acid molecule. In another embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the light chain or an antigen-binding portion thereof of a β-cadherin antibody, and expressing the nucleic acid molecule. In a more preferred method, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule 20 encoding the heavy chain or an antigen-binding portion thereof and an isolated nucleic acid molecule encoding the light chain or the antigen-binding portion thereof of a P cadherin antibody of the invention, and expressing the nucleic acid molecules. The gene therapy method may also include the step of administering another therapeutic agent, such as one of the agents previously discussed in connection with combination therapy.

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any way.

Examples In the following examples and compositions, "BSA" means bovine serum albumin; "EDTA" means ethylenediametetraacetic acid; "DMSO" means dimethyl sulfoxide; 108 means "MOPS" 3- (N-morpholino) propane sulfonic acid; "MES" means 2- (N-morpholino) ethanesulfonic acid; "PBS" means phosphate buffered physiological saline; "dPBS" means Dul-5 beccos with phosphate buffered saline; "HEMA" means 2-hydroxyethyl methacrylate; "DMEM" means Dulbecco's modified eagle's medium; "FBS" means fetal bovine serum; "NEAA" means non-essential amino acids; "HEPES" means N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulfonic acid; and "DMF" means dimethylformamide.

Example 1: Screening an scFv phage display library

Recombinant human β-cadherin (R&D Systems 861-PC-15 100) was used as the antigen for screening an scFv phage display library. Large scFv libraries of human antibodies, cloned into a phagemid vector, were used for the selections (Vaughan, T. J. et al., Nat. Biotech. 14: 309-314 (1996)). ScFv that recognized P-20 cadherin were isolated from the phage display libraries in a series of repeated selection cycles in recombinant human P-cadherin and confluent monolayers of HCT116 cells expressing P-cadherin. Briefly, after incubation with the library, binding phage was recovered from β-cadherin and unbound phage was washed away. Bound phage was then rescued as described in Vaughan, T.J. et al., Nat. Biotech. 14: 309-314 (1996), and the selection process was repeated.

A representative portion of the clones from the yield of the selection rounds was subjected to an enzyme-linked immunoabsorption assay [enzyme-linked immunosorbent assay] (ELISA) at the phage to test for binding to β-cadherin, essentially as described in Vaughan, TJ et al., Nat. Biotech. 35, 14: 309-314 (1996). Two different antigens were used in the ELISA: recombinant human β-cadherin (R&D Systems) and confluent monolayers of A431 cells expressing β-109 cadherlin. ELISA positive clones were DNA sequenced as described in Vaughan, T.J. et al., Nat. Biotech. 14: 309-314 (1996) and in Osbourn, J.K. et al., Immunotechnology 3: 293-5 302 (1998). Unique ELISA positive clones were converted to whole IgG molecules and tested for their ability to neutralize β-cadherin in the β-cadherin-dependent adhesion assay described in Example 4. Based on the results of this screening, the anti- 10 body 129-lc4 (IC50 of 1-3 μΜ in the A431 adhesion assay) was chosen as the lead maternal descent for further optimization.

Example 2: Guideline optimization

Phage display libraries derived from 129-1c4 were created by oligonucleotide-directed mutagenesis of CDR3 regions of the antibody variable heavy (VH) and variable light (VL) chain. The libraries were constructed using standard molecular biology techniques as described in Clackson and Lowman, Phage Display - A Practical Approach (Oxford University Press, 2004). Affinity-based selections were made whereby, after incubation with the library, the recombinant human β-cadherin (R&D Systems) was captured by protein G coated paramagnetic beads (Dynal 100.03), and bound phage were recovered by magnetic separation while unbound complexes were washed away. The selection process was repeated with decreasing concentrations of recombinant human β-cadherin (25 nM to 10 pM in 4 rounds) present during the selection. In addition, selection yields from the VH-CDR3 and VL-CDR3 libraries were recombined into further phage display libraries and selected in two more rounds of affinity-based selection. Representative portions of the 35 clones derived from the yields of the selection rounds were screened as scFv in the 129-1π4 epitope competition assay, as described in Example 8.

Example 3: β-cadherin-dependent adhesion determination

The following protocol was used to determine ICB0 values in a β-cadherin-dependent adhesion assay using various optimized scFvs that were converted to IgG during the optimization phase of antibody discovery, as described above in Example 2. The average measured IC50 values for these antibodies are shown in Table 4 below.

Recombinant human β-cadherin Fc (R & D-cat. 861-PC) was returned to its normal state to a concentration of 1 mg / ml 24 hours prior to use with 2 mM CaCl 2 in Mili-Q water and at 4 ° C stored. A431 cells were cultured and prepared as follows. A431 cells (ECACC No. 85090402) were routinely grown in Nunc triple culture flasks (3 x 175 cm 2 area) in minimal essential medium (MEM) (Invitrogen cat. 31095), containing 10% fetal bovine serum (Invitrogen- cat. 20 10100-147) and 1% non-essential amino acids (Invitrogen cat. 11140-035). The cultured cells were approximately 80% confluent at the time of harvest for use in the assay. To protect against possible passage-related effects, the cells were routinely used between passage 4 and 8, and harvested after either 48 or 72 hours of culture. A431 cells were harvested with 0.25% trypsin / 1 mM EDTA (Gibco-cat. 25200-056) for a time exactly enough for the cells to separate (7-10 min), and then immediately diluted in tissue culture media up to a density of about 2 x 10 6 cells / ml. The A431 cells were then centrifuged (1200 rpm), resuspended in assay buffer (Hanks' balanced salt solution (without Mg2 + and Ca2 +, without phenol red) (Invitrogen cat. 14175-053) supplemented to a final CaCl2-35 concentration of 1 mM), centrifuged and resuspended in assay buffer, again at a final density of 2 x 10 6 cells / ml.

Ill

The preparation of serial dilutions of

IgG and pre-incubation with A431 cells were performed as follows. 180 μΐ of each test IgG, or part thereof, was supplemented with 20 μΐ assay buffer containing 10% BSA (Sig-5 ma. Cat. A-9576), whereby the BSA concentration was normalized to 1%. An anti-mouse / human β-cadherin reference polyclonal antibody (R & D cat. AF-761) was initially resuspended in MilliQ water, yielding a stock of 1 mg / ml. 40 μΐ 10 of this stock solution was then further diluted in 140 μΐ assay buffer. 20 μΐ assay buffer containing 10% BSA was then added, yielding 200 μΐ at 0.2 mg / ml antibody and 0.1% BSA. Serial dilutions were prepared in duplicate by first adding 2 x 90 μΐ AF-761 polyclonal antibody or test IgG to column 1 of a Greiner 96-well polypropylene dilution plate (Greiner cat. 780271). 60 μΐ assay buffer was then added to columns 2-11. A dilution of 30 μΐ to 60 μΐ 20 (1: 3) was then prepared over the plate from left to right of columns 1-11. 60 μΐ assay buffer alone was then added to wells 12 A-D to define the maximum adhesion. Minimum adhesion was defined by adding 60 μΐ 25 mM 25 EDTA (in assay buffer) to wells 12 E-H. 60 μΐ A431 cell suspension (2 x 10 6 cells / ml in assay buffer prepared as outlined above) was then added to all wells, and after movement, the plates were preincubated for 1 h at 37 ° C.

In parallel with the preparation of serial dilutions of test IgG and preincubation with A431 cells, assay plates coated with β-cadherin were prepared as follows. Recombinant human β-cadherin Fc was diluted in coating buffer (PBS without Mg2 + and Ca2 + Invitrogen cat. 14190-094) to a concentration of 10 pg / ml, and spread on 96-well Fluoronunc assay plates (Nunc cat. 437958) (100 μΐ / well). The plates were then incubated for 1 hour and 30 µm at room temperature. The plates were then washed 3 times with PBS using a Tecan 96 plate washer. 200 μΐ / well assay buffer was then added for blocking and the 5 plates were incubated for an additional 1 h at room temperature. The plates were then washed 3 times with PBS as previously described.

100 μΐ / well of the pre-incubated IgG / A431 material was then transferred from the 96-well Greiner dilution plates 10 to the P-cadherin coated assay plates. At the time of the transfer, the IgG / A431 material was mixed by pipetting to ensure that the cells were homogeneous. The adhesion process was then allowed to take place by incubating the assay plates for 30-45 minutes at 37 ° C. At the end of the incubation, non-adherent cells were removed by gently aspirating the media from the plates and refilling the wells with cell wash buffer (Hanks' balanced saline solution (without Mg 2+ and Ca 2+ and without phenol -red - Invitrogen cat. 14175-053), supplemented to a final concentration of 1 mM CaCl 2). The plates were then inverted for 15 minutes in a cell wash buffer bath to remove residual non-adherent cells. At the end of this incubation, the contents of the wells were carefully aspirated.

The quantification of adherent cells was performed as follows. Adherent cells were detected by the addition of 100 μΐ / well of combined lysis / alkaline phosphatase detection reagent (diethanolamine substrate buffer, 5X concentrate (Pierce cat. 34064), diluted 1: 5 with water, and then one PNPP 15 mg tablet (Sigma cat. N-2640) was dissolved per 25 ml of 1X solution), followed by incubation for 30-60 minutes at 37 ° C. The reaction was then stopped by the addition of 1 M NaOH (50 μΐ / well), trying to produce a maximum OD value of about 0.8 in the absence of 113 inhibition. The absorbance at 405 nm was then measured using a standard plate reader.

The results were then analyzed as follows. While column 12, wells AD as 100% adhesion and column 12, wells EH were taken as 0% adhesion, the raw data was first converted to% adhesion values as follows:% adhesion = {(value-min adhesion) / (max -min adhesion)} * 100 The% adhesion versus the IgG inhibitor concentration was then plotted and the IC 50 values were determined using Prism software. Where partial inhibition was observed, the IC 50 is referred to as the concentration of IgG that gives a true 50% inhibition from the center of the curve. IC50 values are reported in Table 4.

Table 4 2 0 __

IgG A431 adhesion assay, average IC 50 (nM) (n = 3) 194- e06__0.162_ 194 -a02__0.217_ 194 -b09__0.229_ 195-ell__0.114_ 194-g09__0.158_ 196-h02 ___ 0.148_ 194-eQ1__0.147_ _196-d10_0.080_ 196-g03_0.149_ 196-e06_0.117 _ 195-a09_0.114_ 198-a09_0.097_ 200-h06 _0.168_ 1X4

Two separate A431 adhesion assays were performed to several germinated, optimized

IgGs from the 129-1c4 lineage to be tested in comparison with their non-germinated equivalents. For these experiments it was necessary to switch to a new batch of β-cadherin (CFR-134041), which appears to be associated with slightly elevated lesson values compared to other data. Average data for various germinated IgGs from the two experiments are shown in Table 5. As noted earlier, g-194-b09 refers to the germinated version of 194-b09, and so on.

15

Table 5

IgO A431 adhesion assay, average IC50 (nM) (n = 2) g-194 -b09__0.77_ 194-b09__2.10_ g-194 -g09__1.00_ 194-g09__0.73_g-196 -g03__1.05_ 196 -g03 0 39_ g-194-e06__0.77_ 194-e06__0.46_ g-195-ell__ 0.87_ 195-ell__0.97 _ g-200-h06__ 1.31_200 -h06 0.63_ Example 4; _ P-cadherin-dependent cell aggregation - determination

The following protocol was used to determine IC50 values in a P-cadherin-dependent aggregation assay using various optimized scFvs that were converted to IgG during the optimization phase of antibody discovery, as described above in Example 2. Because P- Cadherin overexpressing cell lines form tightly cellular aggregates. When placed in suspension culture, cell aggregation can be measured in the presence of β-cadherin antibodies that disrupt cellular aggregation. The average measured IC 50 values for various antibodies are shown below in Table 6.

Plate preparation was performed as follows. Each 96-well assay plate was coated with 50 μΐ polyHEMA (12 mg / ml in 90% ethanol, 10% methanol) and then evaporated to overnight for 6 hours, followed by washing with 3 x 100 μΐ sterile H 2 O before use. Cells were then cultured as follows. Cells derived from the human cell line SW480 (stably expressing β-cadherin G418r) were passed through in full growth medium (qs DMEM (inVitrogen 11995-065), 10% FBS (Omega Scientific FB-02), 1: 100 NEAA (InVitrogen 11140-050), 1: 100 sodium pyruvate (InVitrogen 11360-070), 1: 100 glutamine (InVitrogen 25030-051), 1: 100 penicillin / streptolysin (InVitrogen 15070-063), 1: 100, geneticin 50 mg / ml (Invitrogen 10131-035)) + G418 (500 μg / ml), then separated 1: 3-4 twice a week. Cultures were then frozen in growth medium + 10% DMSO.

On Day 1, the SW480: pCAD cells and the control SW4 8 0: pCLNX (control vector stable) were inoculated at 5 x 10 6 cells / 100 mm dish, at a dilution of no more than 1: 3. The cells were then allowed to grow for 48 hours in culture. Each 100 mm dish provided approximately 10 x 106 cells, or enough for 2 x 96-well plates.

On Day 3, the medium was removed, followed by washing with dPBS (Dulbecco's PBS (InVitrogen 14040-133)), after which the cells were trypsinized in 3 ml / 100 mm dish. Neutralization was then performed after release with two volumes (6 ml) of full growth medium. The plate was then washed three times using a 10 ml pipette, disrupting the clusters. Cells were then counted and a pellet 5 was obtained using a Beckman centrifuge at 1000 rpm for 5 minutes. The medium was then aspirated, the pellet first resuspended in <1 ml of complete growth medium by swirling with the fingers, then with a pOOO pipette, then the cell concentration was normalized to 1.3 M / ml. Dispersion of single cell was assured by microscopy.

A reagent plate was then prepared by blocking a 96-well plate with dPBS and 5% FBS for 30 minutes. The plate was then washed using 1 x 100 μΐ dPBS, followed by aspiration and tapping to dry. A dilution series of test IgG was prepared with dPBS, using 4x [IgG] concentration, enough for 3 wells of the treatment plate in one well of the 96 well. 40,000 cells in 30 μΐ / well were transferred to a washed 96-well polyar HEMA coated Costar3590 non-tissue culture plate (Corning 3590). 10 μΐ of reagent was then transferred to each well of the 96-well plate. Samples were run in triplicate per treatment using a pipette with 8 channels. Incubation then occurred at 250 rpm, shaken, overnight (16-18 h) in a humidified incubator at 37 ° C, 5% CO 2.

3 0 On day 4, 4 0 μΐ of the shaken cells were transferred

to a 96-well polylysine-coated plate (BioCoat-96-well polylysine-coated plate: BD

356516). The wells were then rinsed with 60 μΐ full growth medium, the plate was shaken by tapping, and transferred to the polylysine coated plate. If necessary, an additional wash of 50 μΐ was carried out. Incubation followed for 60 minutes in 117 a humidified incubator of 37 ° C, 5% CO 2. Care was taken in this step that all cells were transferred quantitatively, as gently as possible, without excessive pipetting. Cells were then fixed by adding 100 μΐ fixer solution (7.4% formaldehyde (37% wt / vol. - Sigma F15587)) in a fume cupboard, followed by incubation for> 30 minutes at room temperature.

To wash the cells, the liquid was then poured into a collection beaker or dish and tapped to remove residual liquid, and gently tapped on a tissue. 100 μΐ dPBS per well was then applied for washing, followed by 15 min incubation. The cells were then stained by transfer as above and applying 100 μΐ Hoechst (1 μg / ml Hoechst in dPBS - Hoechst 10 mg / ml, Molecular Probes 33342), followed by incubation for 30 minutes. The cells were then washed twice, leaving the remaining 100 μΐ dPBS in the well for microscopy.

The number of aggregated objects per well was then measured (Cellomics) and an average object count (with test IgG) was compared with that of IgG (eg Gt anti-β-cadherin, R&D Systems AF761) or medium single control. The object count versus the IgG inhibitor concentration was then plotted and the IC50 values were determined. The IC50 values for various antibodies of the present invention are reported in Table 6.

30

Table 6

IgG SW48 0 aggregation assay, average IC5B (nM) _ 194-eOg__0 / 7_ 194-a02__ 1 ^ 1_ g-194 -b09 0_ ^ 9_ iiö g-195-ell__2.2 g-194 -g09__2 / S_ 196-h02__3 / 2_ 194- eOl__1 / 5_ 196-dlO__1 ^ 5_ g-196-g03__0_j_9_ 196-e06__1 ^ 9_ 195-a09__1 / 7_ 198 -a09__2_j_9__ g-200 -h06__4 / 7_ 129-1C4 35_

Example 5 P-cadherin-dependent spheroid disruption assay The following spheroid disruption assay is a variation of the aggregation assay (described in Example 4) in which the cell aggregates are formed overnight prior to the addition of P-cadherin and control antibodies. Test reagents are then added for an additional 24 hours before the analysis.

Plate preparation was performed in the following manner. Each 96-well assay plate was coated with 50 μΐ polyHEMA (12 mg / ml in 90% ethanol, 10% methanol) and then evaporated to overnight for 6 hours, followed by washing with 3 x 100 μΐ sterile H 2 O before use. Cells were then cultured as follows. Cells derived from the human cell line SW480 (stably expressing β-cadherin G418r) were seeded in full growth medium (qs DMEM (InVitrogen 11995-065), 10% FBS (Omega Scientific FB-02), 1: 100 NEAA (InVitrogen 11140-050), 1: 100 sodium pyruvate (InVitrogen 11360-070), 1: 100 glutamine (InVitrogen 25030-051), 1: 100 penicillin / streptolysin (InVitrogen 15070-063), 1: 100, geneticin 50 mg / ml (Invitrogen 10131-035)) + G418 (500 μg / ml), then separated 1: 3-4 twice a week. Cultures were then frozen in growth medium + 10% DMSO.

119

On Day 1, the SW480: pCAD cells and the control SW480: pCLNX (control vector stable) were inoculated at 5 x 10 6 cells / 100 mm dish, at a dilution of no more than 1: 3. The cells were then allowed to grow for 48 hours in culture. Each 100 mm dish provided approximately 10 x 106 cells, or enough for 2 x 96-well plates.

On Day 3, the medium was removed, followed by washing with dPBS (Dulbecco's PBS (InVitrogen 14040-133)), after which the cells were trypsinized in 3 ml / 100 mm dish. Neutralization was then performed after release with two volumes (6 ml) of full growth medium. The plate was then washed three times using a 10 ml pipette, disrupting the clusters. Cells were then counted and a pellet was obtained using a Beckman centrifuge at 1000 rpm for 5 minutes. The medium was then aspirated, the pellet first resuspended in <1 ml of full growth medium by swirling with the fingers, then with a pOOO pipette, then the cell concentration was normalized to 1.0 x 106 / ml. Dispersion of single cell was assured by micröscopy.

40,000 cells in 40 µl / well were transferred to a washed 96-well polyar HEMA coated Costar3590 non-tissue culture plate (Corning 3590). Incubation then took place at 250 rpm, shaken, overnight (16-18 h) in a humidified incubator at 37 ° C, 5% CO 2.

On day 4, a reagent plate was then prepared by blocking a 96-well plate with dPBS and 5% FBS for 30 minutes. The plate was then washed using 1 x 100 μΐ dPBS, followed by aspiration and tapping to dry. A dilution series of test IgG was prepared with dPBS, using 5x [IgG] concentration, enough for 3 wells of the treatment plate in one well of the 96-well plate. 10 μΐ reagent was then transferred to each well 120 µl of the 96-well plate. Samples were run in triplicate per treatment using an 8-channel pipette. Incubation then occurred at 250 rpm, shaken, in a humidified incubator at 37 ° C, 5% CO 2 (20-24 h).

On day 5, 50 μΐ of the shaken cells were transferred to a 96-well polylysine-coated plate (BioCoat-96-well polylysine-coated plate: BD

356516). The wells were then rinsed with 50 μΐ full growth medium, the plate was shaken by tapping, and transferred to the polylysine coated plate. If necessary, an additional wash of 50 μΐ was carried out. Incubation followed for 60 minutes in a humidified incubator of 37 ° C, 5% CO2. In this step, care was taken that all cells were transferred quantitatively, as gently as possible, without excessive pipetting. Cells were then fixed by adding 100 μΐ fixer solution (7.4% formaldehyde (37% wt / vol. - Sigma F15587)) in a fume cupboard, followed by incubation for> 30 minutes at room temperature.

To wash the cells, the liquid was then poured into a collection beaker or dish and tapped to remove residual liquid, and gently tapped on a tissue. 100 μΐ dPBS per well was then applied for washing, followed by 15 min incubation. The cells were then stained by transfer as above and applying 100 μΐ Hoechst (1 μg / ml Hoechst in dPBS 30 - Hoechst 10 mg / ml, Molecular Probes 33342), followed by incubation for 30 minutes. The cells were then washed twice, leaving the remaining 100 μΐ dPBS in the well for microscopy. The number of aggregated objects per well was then measured (Cell 35 lomics) and an average object count (with test igG) was compared with that of IgG (eg Gt anti-β-cadherin, RScD Systems AF761) or medium alone. check. The object 121 count versus the concentration of IgG inhibitor can be plotted to determine the IC 50 values. Alternatively, the object count was expressed as -fold disruption vs. control at one defined concentration, as shown in Table 7.

122

Table 7

IgG SW480 spheroid dislocation assay, x-fold increased disruption vs.

__ check at 5 nM_ 194 -e06__10_ 194 -a02__10_ g-194 -b09__ 10_ g-195-ell ___ 7_, g-194 -g09__14_ 196-h02__10_ 194-eQl__16_ 196-dlO__ 10_ g-196 -g03__ 13_ 196-e06__ 10_ 195-a09__10_ 198-a09__13_ g-200 -h06__7_ 129-lc4 _4_ Example 6: Measurement of Kn and Kaf of β-cadherin antibodies

Dimensions of measurement (Κβ and Kaf) of scFv single chain β-cadherin antibodies by surface plasmon resonance using the BIACORE ™ 3000 instrument were implemented using the manufacturer's protocols as follows.

To perform kinetic analyzes, recombinant human β-cadherin / Fc fusion protein (hCad / Fc) and mouse β-cadherin / Fc fusion protein (mCad / Fc) were immobilized in separate flow cells of a CM5 BIACORE ™ sensor chip using routine amine coupling. Surfaces were prepared using 10 mM acetate buffer, pH 4.5 with 2.0 mM CaCl 2 as the immobilization buffer, and protein densities of 5800 and 1600 RU reached 123 for the hCad / Fc and mCad / Fc, respectively. fusion proteins. Deactivation of N-hydroxysuccinimide esters that had not reacted was performed using 1 M ethanolamine hydrogen chloride, pH 8.5. An activated / deactivated blank surface was used as a control surface. ScFv antibody samples in running buffer were prepared at concentrations ranging from 200 to 0.78 nM (a 0 nM solution comprising only running buffer was included as a zero reference). The samples were randomized and injected through the flow cells each in triplicate for 1 minute using HBS-P (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Surfactant P20) with 2.0 mM CaCl 2 as a running buffer. A flow rate of 25 μΐ / min was used to determine finite constants. The dissociation of the antibody was checked for 5 minutes, the surface was regenerated by means of a 12-second injection of 10 mM glycine HCl, pH 1.5 (25 μΐ / min). The raw data was processed using the Scrubber (© BioLogic Software) software package and analyzed using the CLAMP (© BioLogic Software) software package. The data were made globally suitable for a simple 1: 1 Langmuir binding model.

Table 8 lists affinity constants for the single-chain anti-β-cadherin antibodies of the present invention: Table 8 scFv hCad / Fc, KB tiCad./Fc, Kaf mCad / Fc, Κ „mCad / Fc, Kat __ (nM) __ (1 / s) __ (nM) __ (1 / s) 194 -b0 9__4JD__1.9 x 10 ~ ° 3__11__3.6 X 10 3 194-g09__2 ^ 6__1.6 x 10 03__1_J3__8.1 x 10'4 196- g03 1.1.07 x 10 04 04.76__4.7 X 10 * 124

Example 7: Determining the selectivity of β-cadherin antibodies

The following protocol was used to determine the selectivity of different antibodies for β-cadherin over E-cadherin.

Recombinant human β-cadherin (R&D Systems 861-PC-100) and recombinant human E-cadherin (R&D Systems 648-EC-100) were coated at 1 μg / ml in PBS + 0.5 mM CaCl 2 at 10 wells of Exiqon- protein immobilizer plates (VWR International). Sample IgG1s were blocked in 3% Marvel / PBS + 0.5 mM CaCl2 for 1 hour before being titrated from 50 nM (7.5 μg / ml) to 0.64 nM (0.09.6 μg / ml), and added in duplicate to wells coated with the two different antigens. After equilibrating overnight at 4 ° C, the plates were washed three times with 1 x PBS / 0.1% Tween + 0.5 mM CaCl 2, then three times with 1 x PBS + 0.5 mM CaCl 2. 50 μΐ anti-human Fab peroxidase conjugate, diluted 1: 5000 in 3% Marvel / PBS + 0.5 mM CaCl 2, was then added to each well and allowed to equilibrate for 1 hour at room temperature. The plates were washed three times with 1 x PBS / 0.1% Tween + 0.5 mM CaCl 2 and three times with 1 x PBS + 0.5 mM CaCl 2. 50 μΐ 3.3 l, 5.5'-tetramethylbenzidine (TMB; Sigma) was added to each 25 well and reactions were allowed to develop for 20 minutes before being stopped by the addition of 25 μΐ / well 0, 5 M H 2 SO 4. After reading the absorbance values at 450 nm, the data were analyzed using Graphpad Prism software for calculating relative I 2 values for each antibody with regard to binding to β-cadherin and E-cadherin. The resulting data is summarized in Table 9.

35 125

Table 9

IgG β-cadherin, E-cadherin, KD (E) / KD / (P) __K „(pM) __ K„ (pM) ___ 194 -a02__116__682__6_ g-194 -b09__788__2156__3_ g-194 -g09__2602__No binding __> 100__112 -201_201_201 -201 -201 -201 -201 -201 -201 -201 -201 -201 -201 -201_201 -201 -203 -194-e06__191__794_ 4_ 195 -a09__120__11516 ____ 96_ 194 -e06__56__264__5_ 196-dlO ___ 45_ 63__1 ^ 4_ 196 -e06__62__187__3_ g-196-g03__449__7513__17_ 196-h02__102__9_200_bg_fbg_fbfgb_2008_bbfgb_fbfgb_fbfgb_fbfgb_fbfgb_fbfgb_fbfgb_fbfgfcfcfbfgfbfcfbfcffbfcffbfcffbfcfbfbfcfbfbfbfbfbfbfbfbfbfbfbfbfbfbfbfbffpfgfbfbfpffbffpffbffpffbffpffbffpffbfbfbfpfpff to.

The following protocol was used to measure IC50 values in an epitope competence assay to measure the ability of alternative scFvs within a parent descent to displace the parent IgG of native β-cadherin on the surface of A431- cells. The amount of bound biotinylated parent IgG in the presence of inhibitory scFv is detected with europium streptavidin and DELFIA quantification. The measured lesson values for various scFvs are shown in Table 15.

A431 cells (ECACC No. 85090402), grown according to standard methods, then harvested at approximately 80% confluence, were inoculated at 2.5 x 104 cells per well on the day prior to use in collagen-coated Beckton Dickenson (BD-) cat. 6407) plates with 96 wells. The plates were then washed 3 times by immersion in a PBS buffer (Gibco cat. 14190-094, without calcium, magnesium and sodium bicarbonate) - 126 reservoir, before adding 200 μΐ per well of block buffer (PBS, Gibco cat) 14190-094, plus 3% Marvel (Premier International Foods Ltd.)), and incubated for 2 hours at room temperature. The plates were then washed 3 times with PBS as mentioned above.

For both high-throughput screening and for characterizing the IC50, scFv / IgG materials were first prepared in Greiner dilution plates (Greiner-polypropylene plates with 96 wells (Greiner-cat. 780271)) in a total volume of 60 μΐ assay buffer. Then 50 μ was directly transferred from the co-Greiner plate to the assay plate, and the binding reaction was allowed to take place at room temperature for 2 h and 30 min. At the end of the binding reaction, the plates were washed 3 times in PBS, before the addition of europium-labeled streptavidin (Perkin Elmer cat. 1244-360, dilution of 1: 1000 in DELFIA assay buffer (Perkin Elmer cat) 4002-0010)), and incubated for a further 1 hour at room temperature.

The plates were then washed 7 times with DELFIA wash buffer (Perkin Elmer cat. 4010-0010) by repeatedly immersing the plates in a buffer reservoir. Finally, after adding a DELFIA amplifier (Perkin Elmer cat. 4001-0010 - 100 25 μΐ / well), the plates were read using a standard DELFIA protocol in a compatible reader (eg Wallac or Envision) .

Greiner dilution plate attachment - HTS

Conditions for high throughput screening (high throughput screening) (HTS) were configured differently depending on the stage of optimization. For HTS of yields from the individual VH and VL chain optimization, the end [peri-prep] was set at 12.5%, so that the parent scFv yielded partial inhibition. For the later HTS of yields from the VH: VL recombination libraries, the end-peri-prep concentration was reduced to 1.7%, and under these conditions, the VH-optimized measure-scFv (TOP-108-) C01) partial inhibition. The optimization of peri-prep concentration such that the relevant measure scFv yielded partial inhibition left a window in which improved clones can be identified.

To achieve these final scFv peri-prep concentrations, the following procedure was used: i) Using a Cybiwell instrument 10, the required volume (see below) peri-prep sample material was transferred from a sample plate with deep wells to a Greiner dilution plate in columns 1-11.

[End-peri-prep] Transfer volume 15 [12.5%] = 7.5 μΐ [1.7%] = 10.0 μΐ (from 1:10 diluted peri-prep) (1:10 pre-diluted peri-prep was prepared by transferring 10 μΐ undiluted 20 peri-prep from the sample plate to a Greiner dilution plate containing 90 μΐ assay buffer (using Cybiwell).

ii) The volume in columns 1-11 was then supplemented to 30 µm by the addition of an appropriate volume assay buffer.

iii) 30 μΐ assay buffer was then added to column 12 (A-D) (total binding wells).

iv) 30 μΐ of an excess of unlabelled parent IgG (129-1c4) in assay buffer was added to column 12 (E-H) to define piet-specific binding. This was added at a concentration of 1000 nM, yielding a final concentration of 500 nM.

v) The 129-lc4-IgG was biotinylated using the water-insoluble reagent EZ-1ink-NHS-LC-Biotin (Perbio / Pierce product no.

21336). The IgG solution was supplemented by 129 by adding a 1/10 * volume of 1 M NaHCO 3 and a 1/10 * volume of dimethylformamide. The EZ-link-NHS-LC-Biotin reagent was dissolved in DMF and then added at a five-fold molar excess over IgG, and the reaction was allowed to take place at room temperature for 20 minutes. The biotinylated IgG was then stabilized by the addition of BSA (0.1%). 30 μΐ 10 biotinylated mother 129-lc4 IgG in assay buffer was then added to all wells, yielding a final concentration of 1.5 nM in the final volume of 60 μΐ (ie europium-labeled IgG added at 2x end-15 concentration).

vi) After mixing the Greiner plate contents by motion, 50 μΐ was transferred directly to the assay plate, and the binding reaction was allowed to take place at room temperature for 2 h and 30 min (as previously described).

Greiner dilution plate setup - giving a character sketch of the ICS0 i) 30 μΐ / well assay buffer was added to 2 columns 2-11 and wells 12A to 12D in standard -Greiner dilution plates.

ii) 30 μΐ of an excess of unlabelled 129-lc4 parent IgG in assay buffer was added to column 12 (E-H) to define non-specific binding. This must be added at a concentration of 1000 nM, which results in a final concentration of 500 nM.

iii) To column 1, 2 x 45 μΐ of each undiluted 35 scFv-His-prep was added such that 4 double 11-point IC50 titrations could be set up per 96-well assay plate. Duplicate 1: 3 serial dilutions were then performed by taking 15 μΐ from column 1 and mixing in column 2, followed by taking 15 μΐ from column 2 and mixing in column 3, etc. After mixing in column 11, 15 μΐ was removed (ie leaving a final volume of 30 μΐ).

iv) 30 μΐ biotinylated 129-lc4 parent IgG in assay buffer was then added to all wells, yielding a final concentration of 1.5 nM in the final volume of 60 μΐ (ie europium-labeled IgG added at 2x final concentration ).

V) After mixing the Greiner plate contents by movement, 50 µl was then immediately transferred to the assay plate, and the binding reaction was allowed to take place at room temperature for 2 h and 30 min, as previously discussed.

For HTS, scPv in a 96-well format as a crude extract from the bacterial periplasm (scFv-peri-prep) in a base buffer containing MOPS / EDTA / Sorbitol, pH 7.4 (MES, pH 7.4), expressed. To give a character sketch of the IC 50, scFv were expressed in a purified form of lower throughput, using the scFv-His affinity purification label (scFv-His-prep) in a PBS base buffer.

For both HTS and IC50 analysis, the raw data was first converted to% binding data, according to the following equation:% binding = {(value-non-specific binding) / (total binding-non-specific binding)} * 100 HTS data were then further analyzed using standard Excel templates to identify hits that provide greater inhibition 130 (lower% binding) than the relevant scFv measure. For IC50 analysis,% binding data was analyzed using Prism version 4.0 software to adjust the curve. The scFvs are variants of the 129 Ic4 parent IgG, with the VH and V1 CDRS regions mutated randomly, as described in Example 2. VH-CDR3 variant sequences are shown in Figure 1 as SEQ ID NOs : 91 to 256. VL-CDR3 variant sequences are shown in Figure 1 as SEQ ID NOs: 257 to 319. The IC50 values for various scFvs that exhibited improved binding above the parent 129-1c4 IgG are shown in Table 10. Each of the scFv variants of the 129-lc4 mother is identified on the basis of two SEQ ID NOs: a VH-CDR3 and a VL-CDR3. For reference 15, the IC 50 of the parent l29-lc4 IgG was 108.3 nM.

Table 10 131 SEQ ID NOs Va-CDR3 SEQ ID NO: VL-CDR3 IC 50 (nM) 37 40 INF 49 49 261 1778 3 7 43 §767 37 274 8773 37 277 2720 37 287 10.50 37 288 6.67 37 295 0.80 37 297 2.49 37 ~ 299 1.97 37 303 1.47 37 - 3 4 1.10 37 305 9 95 47 2.36 97 47 1.33 113 47 2.06 125 47 0726 131 47 0 35 31 47 2.05 147 47 1.16 165 47 5.11 167 47 0.60 IN8 47 0.43 IN8 47 2.46 27 47 0.54 195 47 1.29 IN87 43 1.58 27 296 1 , 16 63 63 43 2.45 26 4 0 0.76 26 43 0.93 27 309 1 1.36 199 310 2.54 2 0 3 3 2.26 2 0 7 291 2.73 240 3 3.93 2.98 5 All publications, patents and patent applications those cited in this description are incorporated herein by reference as if it was specifically and individually indicated that each individual publication or patent application is to be incorporated by reference. Although the foregoing invention has been described by way of illustration and example in some detail for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto. without departing from the spirit or scope of the appended claims.

1031818

Claims (16)

An antibody or antigen-binding portion thereof that has at least one property selected from the group consisting of: a) has a KD (E) / KD (P) that is greater than or equal to 1.5; b) binds to β-cadherin with a KD of 50 nM or less, as measured by surface plasma resonance; c) a Ka-β for β-cadherin of less than or equal to 0.01 μl, as measured by surface plasma resonance; d) an IC 50 of 50 nM or less, as measured by a P-cadherin-dependent cell adhesion assay; e) an IC 50 of 50 nM or less, as measured by a β-cadherin-dependent cell aggregation assay; f) increases spheroid disruption in a β-cadherin-dependent spheroid disruption assay by a factor of 2 or more, compared to a control sample without IgG; g) competes with an antibody selected from the group consisting of 194-e06; 194-a02; 94-b09; 195 ell; 194 g09; 196-h02; 194-e01; 196 d10; 196 g03; 196-e06; 195-a09; 198-aO9; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4 and g-129-1c4, for binding to β-cadherin; h) cross-competes with an antibody selected from the group consisting of 194-e06; 194-aO2; 194-b09; 195-ell; 194 g09; 196-h02; 194-eOl; 196-dl0; 196 g03; 196-3.e06; 195 aO9; 198-aO9; 2 00-h06; g-194-bO9; g-194-g09; g-1031818 196-g03; g-196-h02; g-194-eO1; g-194-e06; g-129-lc4 and g-129-lc4, to bind to β-cadherin; i) binds to the same epitope of β-cadherin as an antibody selected from the group consisting of 194-106; 194-a02; 94-b09; 195 ell; 194 g09; 196-h02; 194-eO1; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4 and g-129-1c4; j) binds to β-cadherin with substantially the same KD as an antibody selected from the group consisting of 194-e06; 194-a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eO1; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-e06; 129-1c4 and g-129-1c4; and 15 k) binds to β-cadherin with substantially the same Kaf as an antibody selected from the group consisting of 19 4 - 6; 194-a02; 194-b09; 195 ell; 194 g09; 196-h02; 194-eO1; 196 d10; 196 g03; 196-e06; 195-a09; 198-a09; 200-h06; g-194-b09; g-194-g09; g-196-g03; g-196-h02; g-194-e01; g-194-106; 129-1c4 and g-129-1c4. 2. Antibody or antigen-binding portion thereof according to claim 1, comprising a VH domain that is at least 90% identical in amino acid sequence to one of SEQ ID NOs: 13, 320, 321 and 322. The antibody or antigen-binding portion thereof according to claim 1, comprising a VL domain that is at least 90% identical in amino acid sequence to one of SEQ ID NOs: 22, 326, 327 and 328. The antibody or antigen-binding portion thereof of claim 1 selected from the group consisting of: a) an antibody or antigen-binding portion thereof that has a VH domain as set forth in SEQ ID NO: 13, or that differs from SEQ ID NO : 13 with 1 to 10 conservative amino acid substitutions, and comprises a VL domain as set forth in SEQ ID NO: 22, or different from SEQ ID NO: 22 with 1 to 10 conservative amino acid substitutions; b) an antibody or antigen-binding portion thereof that has a VH domain as set forth in SEQ ID NO: 320, or that differs from SEQ ID NO: 320 with 1 to 10 conservative amino acid substitutions, and a VL domain as set forth in SEQ ID NO: 326, or different from SEQ ID NO: 32 with 1 to 10 conservative amino acid substitutions; c) an antibody or antigen-binding portion thereof that has a VH domain as set forth in SEQ ID NO: 321, or that differs from SEQ ID NO: 321 with 1 to 10 conservative amino acid substitutions, and a VL domain as set forth in SEQ ID NO. : 327, or different from SEQ ID NO: 327 with 1 to 10 conservative amino acid substitutions; and d) an antibody or antigen-binding portion thereof that has a VH domain as set forth in SEQ ID NO: 322, or that differs from SEQ ID NO: 322 with 1 to 10 conservative amino acid substitutions, and a VL domain as set forth in SEQ ID NO: 328, or that differs from SEQ ID NO: 328 with 1 to 10 conservative amino acid substitutions. 25 The antibody or antigen-binding portion thereof of claim 1, comprising a VH domain independently selected from one of SEQ ID NOs: 1 to 13 and 320 to 325, or a sequence different from one of SEQ ID NOs: 1: to 13 and 320 to 325 with 1 to 10 conservative amino acid substitutions; and further comprising a VL domain independently selected from one of SEQ ID NOs: 14 to 23 and 326 to 331, or a sequence different from one of SEQ ID NOs: 14 to 23 and 326 to 331 with 1 to 10 conservative amino acid substitutions. The antibody or antigen-binding portion thereof according to claim 1, wherein said antibody or antigen-binding portion is a VH-CDR3 selected from one of SEQ ID NOs: 26 to 37 and 91 to 256, or a sequence different from one of SEQ ID NOs : 26 to 37 and 91 to 256 with 1 or 2 conservative amino acid substitutions. 7. The antibody or antigen-binding portion thereof according to claim 1, wherein said antibody or antigen-binding portion is a VL-CDR3 selected from one of SEQ ID NOs: 40 to 47 and 257 to 319, or a sequence different from one of SEQ ID NOs: 40 to 47 and 257 to 319 with 1 or 2 conservative amino acid substitutions. The antibody or antigen-binding portion thereof of claim 1, wherein said antibody or antigen-binding portion: a) a VH-CDR1 as set out in SEQ ID NO: 24, or a sequence different from SEQ ID NO: 24 with 1 or 2 conservative amino acid substitutions; b) a V 1 -CDR 2 as set forth in SEQ ID NO: 25, or a sequence different from SEQ ID NO: 25 with 1 or 2 conservative amino acid substitutions; and c) a V '-CDR3 selected from one of SEQ ID NOs: 26 to 37 and 91 to 256, or a sequence different from one of SEQ ID NOs: 26 to 37 and 91 to 256 with 1 or 2 conservative amino acid substitutions. The antibody or antigen-binding portion thereof according to claim 1, wherein said antibody or antigen-binding portion: a) a VL-CDR1 as set forth in SEQ ID NO: 38, or a sequence different from SEQ ID NO: 3 by 1 35 or 2 conservative amino acid substitutions; b) a VL-CDR2 as set forth in SEQ ID NO: 39, or a sequence different from SEQ ID NO: 39 with 1 or 2 conservative amino acid substitutions; and c) a VL-CDR3 selected from one of SEQ ID NOs: 40 to 47 and 257 to 319, or a sequence different from one of SEQ ID NOs: 40 to 47 and 257 to 319 with 1 or 2 conservative amino acid substitutions. 10 An antibody or antigen-binding portion thereof, wherein the VH domain is one of SEQ ID NOs: 13, 320, 321, and 322, or a sequence different from one of SEQ ID NOs: 13, 320, 321, and 322 with 1 up to 10 conservative amino acid substitutions; and the VL domain comprises one of SEQ ID NOs: 22, 326, 327 and 328, or differs from one of SEQ ID NOs: 22, 326, 327 and 328 with 1 to 10 conservative amino acid substitutions. The antibody of any one of claims 1 to 10 that is, or is derived from, an IgG, an IgM, an IgE, an IgA, or an IgD molecule. The antibody of claim 11 wherein the IgG 2 is an IgGj wherein the constant region of the heavy chain comprises SEQ ID NO: 344 and wherein the constant region of the light chain comprises SEQ ID NO: 345, provided that the C-terminal lysine residue of SEQ ID NO: 344 is optionally split. 30 A pharmaceutical composition comprising the antibody or antigen-binding portion of any one of claims 1 to 12 and a pharmaceutically acceptable carrier. An isolated nucleic acid molecule comprising a nucleotide sequence as set forth in one of SEQ ID NOs: 80, 89, 332, 333, 334, 338, 339 and 340. The antibody or antigen-binding portion thereof of claim 1, comprising an amino acid sequence of a heavy chain variable region using a human VH-3 family gene. The antibody or antigen-binding portion thereof of claim 1 comprising a first variable domain comprising SEQ ID NO: 321 and a second variable domain comprising SEQ ID NO: 327.