HK1117172B - P-cadherin antibodies - Google Patents

Description

P-cadherin antibodies

This application claims priority to U.S. provisional application No.60/675,311, filed on 26/4/2005, which is incorporated herein by reference in its entirety.

Technical Field

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

Background

Cadherins are a superfamily of transmembrane glycoproteins that regulate cell-cell adhesion during development and tissue homeostasis (Gumbiner J. cell. biol., 148: 399-. The intracellular domain of cadherin interacts with cytoplasmic proteins (e.g., catenin and p120), which form the basis for cadherin attachment to the actin cytoskeleton. Cadherins have five extracellular Ca' s²⁺Bonding ofA domain and a small cytoplasmic domain that is highly conserved in classical cadherins. Classical cadherin family members include P-cadherin, E-cadherin, and N-cadherin. Cell adhesion molecules such as cadherins are thought to play an important role in the cellular communication between cancer cells and metastatic cells (Furukawa, et al, Microcopy Res. technique 38 (4): 343-352 (1997)). P-cadherin is underexpressed in normal adult tissues and is essentially restricted to the basal layer of myoepithelial cells and stratified epithelia (Shimoyama, et al. cancer Res.49: 2128-33 (1989)). P-cadherin is upregulated in inflammatory bowel diseases such as Crohn's disease and colitis (Hardy, et al, Gut 50: 513-519 (2002)). There is now a great deal of evidence that: abnormal P-cadherin expression is associated with cell proliferation and tumors of the colon, breast, lung, thyroid and cervix (Gamallo, Modern Pathology, 14: 650-. Human P-cadherin has been reported to be an antigen recognized by the NCC-CAD-299 monoclonal antibody raised against vulvar epidermoid carcinoma (Shimoyama, et al, Cancer Res., 49: 2128-2133 (1989)). Modulation of P-cadherin-mediated adhesion and intracellular signal transduction is expected to result in decreased proliferation and survival of tumor cells in vivo. Thus, given that P-cadherin appears to have a key role in controlling cell proliferation and development of solid tumors, it would be desirable to generate antibodies against P-cadherin that can provide therapeutic benefit to a variety of cancer patients.

Disclosure of Invention

In one aspect, the invention is a P-cadherin antibody, or an antigen-binding portion thereof, wherein the antibody, or antigen-binding portion thereof, has at least one of the several functional characteristics described in a) through K) below. A) For example, in one embodiment, the antibody, or antigen binding portion thereof, has a binding affinity for E-cadherin (K)_D(E) Comparison against P-cadherin (K)_D(P)) greater binding affinity. In one embodiment, the antibody or antigen binding of the inventionThe sum of the moieties has a K of greater than or equal to 1.5_D(E)/K_D(P) of the reaction mixture. In another embodiment, an antibody or antigen-binding portion thereof of the invention has a K 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 or equal to 100, greater than or equal to 200, greater than or equal to 500, or greater than or equal to 1000_D(E)/K_D(P) of the reaction mixture. Typically, K_D(E)/K_DThere is no upper limit to the value of (P) because K_D(E) The value may be very small, for example 0. However, for practical purposes, K_D(E)/K_DThe upper limit of (P) may be 1x10⁶. For P-cadherins and E-cadherins, such K' s_DThe values can be measured by any technique known to those skilled in the art, for example by ELISA, RIA, flow cytometry or surface plasmon resonance, for example BIACORE^TM。

B) In another embodiment, the antibody or portion thereof has a K of 1000nM or less as measured by surface plasmon resonance_DBinds to P-cadherin. In another embodiment, the antibody or portion thereof has a K of less than 500nM, less than 100nM, less than 50nM, less than 20nM, less than 10nM, less than 1nM, less than 500pM, or less than 100pM, as measured by surface plasmon resonance_DBinds to P-cadherin. Typically, K is not present_DThe lower limit of the value. However, for practical purposes, the lower limit may be assumed to be about 1 pM.

C) In another embodiment, the antibody or portion thereof has less than or equal to 0.01 for P-cadherin as measured by surface plasmon resonance^s-1Clearance rate (k) of_off). For example, in certain embodiments the antibody or portion thereof has less than 0.005 for P-cadherin^s-1Less than 0.004^s-1Less than 0.003^s-1Less than 0.002^s-1Or less than 0.001^s-1K of (a)_off. Typically, k is not present_offThe lower limit of the value. However, for practical purposes, the lower limit may be assumed to be about 1x10^{-7 s-1}。

D) In another embodiment, the P-cadherin antibody or portion thereof has an IC of 100nM or less as measured by a P-cadherin-dependent cell adhesion assay₅₀. In another embodiment, the IC is as determined by a P-cadherin-dependent cell adhesion assay₅₀Less than 50nM, less than 40nM, less than 20nM, less than 10nM, less than 1nM, less than 500pM, less than 200pM, less than 100pM, or less than 10 pM. Typically, there is no IC as determined by a P-cadherin-dependent cell adhesion assay₅₀The lower limit of the value. However, for practical purposes, the lower limit may be assumed to be about 1 pM.

E) In another embodiment, the P-cadherin antibody or portion thereof has an IC of 100nM or less as measured by a P-cadherin-dependent cell aggregation assay₅₀. In another embodiment, the IC is as determined by a P-cadherin-dependent cell aggregation assay₅₀Less than 50nM, less than 40nM, less than 20nM, less than 10nM, less than 1nM, less than 500pM, less than 200pM, less than 100pM, or less than 1 pM. Typically, there is no IC present as determined by a P-cadherin-dependent cell aggregation assay₅₀The lower limit of the value. However, for practical purposes, the lower limit may be assumed to be about 1 pM.

F) In another embodiment, the P-cadherin antibody or portion thereof increases spheroid destruction by a factor of at least 2 in a P-cadherin-dependent spheroid destruction assay as compared to a control sample in the absence of IgG. In another embodiment, the P-cadherin antibody or portion thereof increases spheroid destruction by a factor of at least 3, at least 4, at least 6, at least 10, or at least 15 in a P-cadherin-dependent spheroid destruction assay as compared to a control sample in the absence of IgG.

G) In another embodiment, the P-cadherin antibody or portion thereof is conjugated to a peptide selected from the group consisting of 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and the antibody of g-129-1c4 competes for binding to P-cadherin.

H) In another embodiment, the P-cadherin antibody or portion thereof is conjugated to a peptide selected from the group consisting of 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e06129-1c 4; and antibody g-129-1c4 cross-competed for binding to P-cadherin.

I) In another embodiment, the P-cadherin antibody or portion thereof is conjugated to a peptide selected from the group consisting of 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and the antibody of g-129-1c4 binds to the same epitope of P-cadherin.

J) In another embodiment, the P-cadherin antibody or portion thereof is conjugated to a peptide selected from the group consisting of 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and the antibody of g-129-1c4 with substantially the same K_DBinds to P-cadherin.

K) In another embodiment, the P-cadherin antibody or portion thereof is conjugated to a peptide selected from the group consisting of 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; antibodies to g-129-1c4 have substantially the same k_offBinds to P-cadherin.

Another aspect of the invention is an antibody or antigen-binding portion thereof having at least one of the functional features previously described in a) to K), comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NOs: v of at least 90% identity to any one of 1 to 13 and 320 to 325_HA domain. In one embodiment, said V is_HDomain amino acid sequence and SEQ ID NOs: any one of 1 to 12 and 320 to 325 is at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% identical.

In another embodiment, the antibody or part thereof has at least one of the functional characteristics previously described in A) to K), comprising one V_HDomain of the V_HThe domain is SEQ ID NOs: 1 to 13 and 320 to 325, or a variant thereof which has at least one conservative amino acid substitution with SEQ ID NOs: 1 to 13 and 320 to 325. For example, V_HThe domains are identical to SEQ ID NOs: 1 to 13 and 320 to 325. In another embodiment, any of these conservative amino acid substitutions may occur in the CDR1, CDR2, and/or CDR3 regions.

Another aspect of the invention is an antibody or antigen-binding portion thereof having at least one of the functional features previously described in a) to K), comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NOs: v of at least 90% identity to any one of 14 to 23 and 326 to 331_LA domain. In one embodiment, said V is_LDomain amino acid sequence and SEQ ID NOs: any of 14 to 23 and 326 to 331 are at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% identical.

In another embodiment, the antibody or part thereof has at least one of the functional characteristics described previously in A) to K) and comprises one V_LDomain of the V_LThe domain is SEQ ID NOs: 14 to 23 and 326 to 331, or a variant thereof which has at least one conservative amino acid substitution with seq id NOs: 14 to 23 and 326 to 331. E.g. V_LThe domains are identical to SEQ ID NOs: 14 to 23 and 326 to 331. In another embodiment, any of these conservative amino acid substitutions may occur in the CDR1, CDR2, and/or CDR3 regions.

Another aspect of the invention is an antibody or antigen-binding portion thereof having at least one of the functional characteristics previously described in A) to K), wherein V_LAnd V_HDomains with V of antibodies_LAnd V_HA domain amino acid sequence at least 90% identical, said antibody being selected from 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and g-129-1c 4. For example, V_LAnd V_HDomains with V of antibodies_LAnd V_HA domain amino acid sequence that is at least 91%, 93%, 95%, 97%, 99% or 100% identical, said antibody being selected from 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and g-129-1c 4.

Another aspect of the invention is an antibody or antigen-binding portion thereof selected from the group consisting of: a) comprises the amino acid sequence shown as SEQ ID NO: 1 disclosed in_HDomain and the amino acid sequence as set forth in SEQ ID NO: 14 disclosed in V_LAn antibody or portion thereof of a domain; b) comprises the amino acid sequence shown as SEQ ID NO: 2 disclosed in V_HA domain and a polypeptide as set forth in SEQ ID NO: 14 disclosed in V_LAn antibody or portion thereof of a domain; c) comprises the amino acid sequence shown as SEQ ID NO: 2 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 15 disclosed is a V_LAn antibody or portion thereof of a domain; d) comprises the amino acid sequence shown as SEQ ID NO: 3 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 16 disclosed in V_LAn antibody or portion thereof of a domain; e) comprises the amino acid sequence shown as SEQ ID NO: 4 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 17 of the disclosure_LAn antibody or portion thereof of a domain; f) comprises the amino acid sequence shown as SEQ ID NO: 4 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 23 of the publication V_LAn antibody or portion thereof of a domain; g) comprises the amino acid sequence shown as SEQ ID NO: 5 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 18 of the disclosure_LAntibodies to domainsOr a portion thereof; h) comprises the amino acid sequence shown as SEQ ID NO: 6 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 23 of the publication V_LAn antibody or portion thereof of a domain; i) comprises the amino acid sequence shown as SEQ ID NO: 7 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 23 of the publication V_LAn antibody or portion thereof of a domain; j) comprises the amino acid sequence shown as SEQ ID NO: 8 disclosed in V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 23 of the publication V_LAn antibody or portion thereof of a domain; k) comprises the amino acid sequence shown as SEQ ID NO: 9 of the disclosure_HDomain and the amino acid sequence as set forth in SEQ ID NO: 23 of the publication V_LAn antibody or portion thereof of a domain; 10 comprises the amino acid sequence shown as SEQ ID NO: 10 of the publication V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 19 of the publication V_LAn antibody or portion thereof of a domain; m) comprises the amino acid sequence as set forth in SEQ ID NO: 11 disclosed in_HDomain and the amino acid sequence as set forth in SEQ ID NO: 20 of the disclosure_LAn antibody or portion thereof of a domain; n) comprises the amino acid sequence as set forth in SEQ ID NO: 12 of the publication V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 21 disclosed in V_LAn antibody or portion thereof of a domain; o) comprises the sequence as set forth in SEQ ID NO: 13 of the publication V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 22 disclosed as V_LAn antibody or portion thereof of a domain; p) comprises the sequence as set forth in SEQ ID NO: 320 of the publication_HDomain and the amino acid sequence as set forth in SEQ ID NO: 326 disclosed V_LAn antibody or portion thereof of a domain; q) comprises the sequence as set forth in SEQ ID NO: 321 of the disclosure_HDomain and the amino acid sequence as set forth in SEQ ID NO: 327 of a disclosed V_LAn antibody or portion thereof of a domain; r) comprises the sequence as set forth in SEQ ID NO: 322 disclosed as_HDomain and the amino acid sequence as set forth in SEQ ID NO: 328 discloses a V_LAn antibody or portion thereof of a domain; s) comprises the sequence as set forth in SEQ ID NO: 323 of_HDomain and the amino acid sequence as set forth in SEQ ID NO: 329 publication of V_LAn antibody or portion thereof of a domain; t) comprises the sequence as set forth in SEQ ID NO: 324 disclosed V_HDomain and the amino acid sequence as set forth in SEQ ID NO: 330 disclosed V_LAn antibody or portion thereof of a domain; and u) comprises the sequence as set forth in SEQ ID NO: 325V of publication_HDomain and the amino acid sequence as set forth in SEQ ID NO: 331 published by V_LAn antibody or portion thereof of a domain.

In another embodiment, V is for any antibody or portion thereof as described above in groups a) to u)_HAnd/or V_LThe domains may differ from the particular SEQ ID NOs described herein by at least one conservative amino acid substitution. E.g. V_HAnd/or V_LThe domains may differ from said SEQ ID NOs by 1,2, 3,4, 5,6, 7, 8, 9 or 10 conservative amino acid substitutions. In another embodiment, any of these conservative amino acid substitutions may occur in the CDR1, CDR2, and/or CDR3 regions.

In another embodiment, the invention provides an antibody or antigen-binding portion thereof having at least one of the functional characteristics previously described in A) to K), comprising one V_HDomains and a V_LDomain of the V_HDomains are independently selected from SEQ ID NOs: 1 to 13 and 320 to 325, or to any one of SEQ ID NOs: 1 to 13 and 320 to 325 differ by at least one conservative amino acid substitution; the V is_LDomains are independently selected from SEQ ID NOs: 14 to 23 and 326 to 331 or a variant thereof of SEQ ID NOs: any one of sequences 14 to 23 and 326 to 331 which differ by at least one conservative amino acid substitution. For example, V_HAnd V_LThe domains may each independently be identical to SEQ ID NOs: any of 1 to 13, 320 to 325, 14 to 23, and 326 to 331 differ by 1,2, 3,4, 5,6, 7, 8, 9, or 10 conservative amino acid substitutions.

In another embodiment, the invention provides an antibody or antigen-binding portion thereof having at least one of the functional characteristics previously described in a) to K), wherein said antibody or portion comprises a heavy chain variable region selected from the group consisting of seq id NOs: v of any one of 26 to 37 and 91 to 256_HCDR3, or a variant of SEQ ID NOs: any one of 26 to 37 and 91 to 256 differs by at least one conservative amino acid substitution. For example, V_HCDR3 can be related to SEQ ID NOs: any of 26 to 37 and 91 to 256 differ by 1,2, 3 or 4 conservative amino acid substitutions.

In another embodiment, the invention provides a composition having at least one of the compounds previously described in A) to K)An antibody or antigen-binding portion thereof of the functional characteristics, wherein said antibody or portion comprises an amino acid sequence selected from the group consisting of seq id NOs: v of any one of 40 to 47 and 257 to 319_LCDR3, or a variant of SEQ ID NOs: 40 to 47 and 257 to 319, which differ by at least one conservative amino acid substitution. For example, V_LCDR3 can be related to SEQ ID NOs: any of 40 to 47 and 257 to 319 differ by 1,2, 3 or 4 conservative amino acid substitutions.

In another embodiment, the invention provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises: as shown in SEQ ID NO: v disclosed in 24_HCDR1, or to seq id NO: 24 by at least one conservative amino acid substitution; as shown in SEQ ID NO: v disclosed in 25_HCDR2, or a variant of SEQ ID NO: 25 by at least one conservative amino acid substitution; and independently selected from SEQ ID NOs: v of any one of 26 to 37 and 91 to 256_HCDR3, or a variant of SEQ ID NOs: any one of 26 to 37 and 91 to 256 differs by at least one conservative amino acid substitution. E.g. each V mentioned above_HThe CDR1, CDR2, and CDR3 sequences may differ independently from the respective SEQ ID NOs by 1,2, 3,4, or 5 conservative amino acid substitutions.

In another embodiment, the invention provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises: as shown in SEQ ID NO: 38 of the formula V_LCDR1, or to seq id NO: 38 by at least one conservative amino acid substitution; as shown in SEQ ID NO: 39 of a compound of formula V_LCDR2, or a variant of SEQ ID NO: 39 that differ by at least one conservative amino acid substitution; and independently selected from SEQ ID NOs: v of any one of 40 to 47 and 257 to 319_LCDR3, or a variant of SEQ ID NOs: 40 to 47 and 257 to 319, which differ by at least one conservative amino acid substitution. E.g. each V mentioned above_LThe CDR1, CDR2, and CDR3 sequences may differ independently from the respective SEQ ID NOs by 1,2, 3,4, or 5 conservative amino acid substitutions.

The invention also provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises: as shown in SEQ ID NO: v disclosed in 24_HA CDR 1; as shown in SEQ ID NO: v disclosed in 25_HA CDR 2; selected from the group consisting of SEQ ID NOs: v of any one of 26 to 37 and 91 to 256_HA CDR 3; as shown in SEQ ID NO: 38 of the formula V_LA CDR 1; as shown in SEQ ID NO: 39 of a V_LA CDR 2; and selected from the group consisting of SEQ ID NOs: v of any one of 40 to 47 and 257 to 319_LCDR 3. In another embodiment, each of said V_HAnd V_LThe CDR1, CDR2, and CDR3 sequences may also differ independently from the particular SEQ ID NOs described above by at least one conservative amino acid substitution. For example, each CDR1, CDR2, and CDR3 sequence may differ independently from the particular SEQ ID NOs described above by 1,2, 3,4, or 5 conservative amino acid substitutions.

The invention also provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises an amino acid sequence as found in 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and V in any of the antibodies of g129-1c4_HAnd V_L CDR1、V_HAnd V_LCDR2 and V_HAnd V_L CDR3。

The invention also provides an antibody, or antigen-binding portion thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 20 to 325 or a variant thereof of SEQ ID NOs: any one of V1 to 3 and 20 to 325 differing by 1 to 10 conservative amino acid substitutions_HA domain.

The invention also provides an antibody, or antigen-binding portion thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 23 and 326 to 331 or any of SEQ ID NOs: any one of V14 to 23 and V326 to 331 which differs by 1 to 10 conservative amino acid substitutions_LAnd (4) domain formation.

The invention also provides antibodies, or antigen-binding portions thereof, comprisingA V_HDomains and a V_LDomain of the V_HDomains are independently selected from SEQ ID NOs: 1 to 13 and 320 to 325, or to any one of SEQ ID NOs: 1 to 13 and 320 to 325 differ by 1 to 10 conservative amino acid substitutions; the V is_LDomains are independently selected from SEQ ID NOs: 14 to 23 and 326 to 331 or a variant thereof of SEQ ID NOs: any one of 14 to 23 and 326 to 331 differs by from 1 to 10 conservative amino acid substitutions.

The invention also provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises: as shown in SEQ ID NO: v disclosed in 24_HCDR1, or a variant of SEQ ID NO: 24 sequences that differ by 1 to 4 conservative amino acid substitutions; as shown in SEQ ID NO: v disclosed in 25_HCDR2, or a variant of SEQ ID NO: 25 sequences that differ by 1 to 4 conservative amino acid substitutions; and selected from the group consisting of SEQ ID NOs: v of any one of 26 to 37 and 91 to 256_HCDR3, or to seq id NOs: any one of 26 to 37 and 91 to 256 differs by 1 to 4 conservative amino acid substitutions.

The invention also provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises: as shown in SEQ ID NO: 38 of the formula V_LCDR1, or a variant of SEQ ID NO: 38 by 1 to 4 conservative amino acid substitutions; as shown in SEQ ID NO: 39 of a compound of formula V_LCDR2, or a variant of SEQ ID NO: 39 that differ by 1 to 4 conservative amino acid substitutions; and selected from the group consisting of SEQ ID NOs: v of any one of 40 to 47 and 257 to 319_LCDR3, or to seq id NOs: any one of 40 to 47 and 257 to 319 differ by 1 to 4 conservative amino acid substitutions.

The invention also provides an antibody or antigen-binding portion thereof having at least one of the functional features previously described in a) to K), wherein the antibody or antigen-binding portion comprises: as shown in SEQ ID NO: v disclosed in 48_HFR 1; as shown in SEQ ID NO: v disclosed in 49_HFR 2; selected from the group consisting of SEQ ID NOs: v of any one of 50 to 55_HFR 3; selected from SEQ ID NAnd Os: 56 and 57, V_HFR 4; selected from the group consisting of SEQ ID NOs: v of any one of 58 and 59_LFR 1; selected from the group consisting of SEQ ID NOs: v of any one of 60 to 62_LFR 2; selected from the group consisting of SEQ ID NOs: v of any one of 63 to 66_LFR 3; and as shown in SEQ ID NO: 67 of_LFR 4. In another embodiment, each of said V_HAnd V_LFR1, FR2, FR3 and FR4 sequences may also differ independently from the specific SEQ ID NOs mentioned above by at least one conservative amino acid substitution. For example, each of the FR1, FR2, FR3, and FR4 sequences can differ independently from the respective specific SEQ ID NOs described above by 1,2, 3,4, 5,6, 7, 8, 9, or 10 conservative amino acid substitutions. In yet another embodiment, any one of each of FR1, FR2, FR3 and FR4 sequences may be independently mutated to match the respective germline framework sequence.

Another embodiment of the invention is any of the antibodies described herein, wherein the antibody is an IgG, IgM, IgE, IgA, or IgD molecule, or is derived therefrom. For example, the antibody may be an IgG₁Or IgG₂. For example, in one embodiment, the IgG is an IgG₁Wherein the heavy chain constant region comprises seq id NO: 344, wherein the light chain constant region comprises SEQ ID NO: 345, and SEQ ID NO: 344 is optionally cleaved.

Another embodiment provides any of the antibodies or antigen binding portions described above that are Fab fragments, F (ab')₂Fragment, F_VFragment, Single chain F_VFragment, Single chain V_HFragment, Single chain V_LA fragment, a humanized antibody, a chimeric antibody or a bispecific antibody.

Another embodiment is a derivatized antibody or antigen-binding portion comprising any of the antibodies or portions thereof described herein and at least one additional molecular entity. For example, the at least one additional molecular entity may be another antibody (e.g., a bispecific antibody or diabody), a detection agent, a tag, a cytotoxic agent, an agent, and/or a protein or peptide capable of mediating association of an antibody or antibody moiety with another molecule (e.g., a streptavidin core region or a polyhistidine tag). For example, useful detection agents with which the antibodies or antigen-binding moieties of the invention can be derivatized include fluorescent compounds, including fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonic acid, phycoerythrin, lanthanide phosphors, and the like. The antibody may also be labeled with an enzyme suitable for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. In another embodiment, the antibodies of the invention or portions thereof can also be labeled with biotin, or with a predetermined polymorphic epitope (e.g., leucine zipper pair sequence, binding site of a secondary antibody, metal binding domain, epitope tag) that is recognized by a secondary reporter. In yet another embodiment of the invention, any antibody or portion thereof may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or carbohydrate groups.

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

In some embodiments, the heavy chain C-terminal lysine of any of the P-cadherin antibodies of the present invention is cleaved. In various embodiments of the invention, the heavy and light chains of the P-cadherin antibody may optionally include N-terminal signal sequences. For example, the heavy chain signal sequence may be SEQ ID NO: 346, the light chain signal sequence may be SEQ ID NO: 347.

in another embodiment, the invention relates to any of the antibodies or antigen binding portions thereof described herein, wherein the antibody or antigen binding portion thereof is of human origin.

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

In another embodiment, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the antibodies or antigen-binding portions thereof described herein. A particular embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NOs: 68 to 90 and 332 to 343. The invention also relates to a vector comprising any of the nucleic acid molecules described herein, wherein said vector optionally comprises an expression control sequence operably linked to said nucleic acid molecule.

Another embodiment provides a host cell comprising any of the vectors described herein, or comprising any of the nucleic acid molecules described herein. The invention also provides an isolated cell line that produces any of the antibodies or antigen-binding portions described herein, or that produces a heavy chain or a light chain of any of the antibodies or the antigen-binding portions.

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

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

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

The invention also provides a method for treating abnormal cell growth in a mammal in need thereof, the method comprising the step of administering to the mammal any of the antibodies or antigen-binding portions thereof described herein, or any pharmaceutical composition. The invention also provides a method for treating abnormal cell growth in a mammal in need thereof using an antibody or antigen-binding portion thereof that binds P-cadherin, comprising the step of administering to the mammal an effective amount of any of the nucleic acid molecules described herein under suitable conditions that allow expression of the nucleic acid molecule. In another embodiment, the method of treating abnormal cell growth further comprises administering an amount of one or more substances selected from the group consisting of an anti-neoplastic agent, an anti-angiogenic agent, a signal transduction inhibitor, and an antiproliferative agent, which together are effective to treat the abnormal cell growth. In a specific embodiment, the abnormal cell growth is cancerous.

The invention also provides a method for reducing P-cadherin-dependent cell aggregation, comprising contacting a cell with any of the antibodies or antigen-binding portions thereof described herein or any of the pharmaceutical compositions described herein.

Another aspect of the invention is an antibody, or antigen-binding portion thereof, comprising the use of human V_H-the heavy chain variable region amino acid sequence of a family 3 gene. For example, in one embodiment, human V_H-3 family Gene is V_H-3-23。

Another aspect of the invention provides any of the antibodies or antigen-binding portions thereof described herein, wherein the antibody or antigen-binding portion is a human antibody. In another aspect, the antibody or antigen-binding portion is a human recombinant antibody.

The present invention also provides a method for producing a P-cadherin antibody, or an antigen-binding portion thereof, comprising the steps of: synthesizing a human antibody library on a bacteriophage, screening the library with P-cadherin or an antigenic portion thereof, isolating the bacteriophage that binds to P-cadherin, and obtaining the antibody from the bacteriophage.

Definitions and general techniques

Unless defined otherwise herein, scientific and technical terms used in relation to the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the terminology used in relation to, or the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art.

Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art, and as described in various general and more specific references that are listed and described throughout the present specification. See, e.g., Sambrook, et al.Molecular Cloning：A Laboratory Manual，3rd ed.，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(2000)；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 ManualCold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan, et al,Short Protocols in Protein Science，Wiley，John&sons, inc. (2003), the disclosure of which is incorporated herein by reference. The enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The terms used in relation to analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemistry described herein, or the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery and treatment of patients.

The basic antibody building block is known to comprise tetramers. Each tetramer consists of two identical polymorphic chain pairs, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 120 or more amino acids, which is primarily responsible for antigen recognition. The carboxy terminus of each chain defines a constant region, which is primarily responsible for effector functions. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α or e, and define antibody isotypes as IgM, IgD, IgG, IgA and IgE, respectively. In both light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a region of about 3 or more amino acids "And D' region. General referenceFundamental ImmunologyCh.7(Paul, w., ed., 2nd ed. raven Press, n.y. (1989)) (incorporated herein by reference in its entirety for all purposes). Each heavy chain/light chain pair (V)_HAnd V_L) Forming antibody binding sites. Thus, for example, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are identical.

The variable regions of the heavy and light chains exhibit the same general structure, being relatively conserved Framework Regions (FRs), also known as complementarity determining regions or CDRs, joined by three highly variable regions. The term "variable" refers to the following: certain portions of the variable regions within an antibody differ greatly in sequence and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not equally distributed throughout the variable domain of the antibody, but is concentrated in CDRs that are separated by more highly conserved FRs. The CDRs from each pair of two chains are aligned by the FRs to enable binding to a particular epitope. From N-terminus to C-terminus, both the light and heavy chains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The amino acid assignment for each domain is according to Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J., mol.biol., 196: 901-917 (1987); chothia, et al, Nature 342: 878-883(1989), the disclosure of which is incorporated herein by reference.

Unless otherwise indicated, the following terms shall be understood to have the following meanings:

unless otherwise specifically stated, the term "P-cadherin" refers to human P-cadherin, a member of the classical cadherin family of membrane intrinsic proteins and transmembrane glycoproteins that regulate cell-cell adhesion. The cloning and sequence of human P-cadherin has been reported, for example, in 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 intended to include recombinant human P-cadherin and recombinant chimeric forms of P-cadherin, which may be prepared by standard recombinant expression methods or commercially available (R & DSsystems 861-PC-100).

Unless specifically stated otherwise, the term "E-cadherin" as used herein refers to human E-cadherin, a member of the classical cadherin family of membrane intrinsic proteins and transmembrane glycoproteins that regulate cell-cell adhesion. E-cadherins are described, for example, in Takeichi, Science, 251: 1451-1455(1991), the disclosure of which is incorporated herein by reference. The term E-cadherin is intended to include recombinant human E-cadherin and recombinant chimeric forms of E-cadherin, which may be prepared by standard recombinant expression methods or commercially available (R & D Systems 861-EC-100).

The term "polypeptide" includes natural or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or multimeric.

The terms "isolated protein," "isolated polypeptide," or "isolated antibody" are proteins, polypeptides, or antibodies that, by virtue of their origin or derivative source, (1) do not have a component with which they naturally associate in their native state, (2) do not contain other proteins from the same species, (3) are expressed by cells from a different species, or (4) do not naturally occur. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell from which it is naturally derived should be "isolated" from the components with which it is naturally associated. Proteins can also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

Examples of isolated antibodies include P-cadherin antibodies that have been affinity purified using P-cadherin, and P-cadherin antibodies synthesized in vitro by cell lines.

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 species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein may typically comprise about 50%, 60%, 70%, 80% or 90% w/w protein sample, more often about 95%, and preferably may be greater than 99% pure. Protein purity can be demonstrated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by staining the gel with dyes well known in the art to visualize a single polypeptide band. For some purposes, higher resolution may be provided by using HPLC or other means well known in the art of purification.

The term "polypeptide fragment" as used herein refers to a polypeptide having an amino-terminal and/or carboxy-terminal deletion, but the remaining amino acid sequence is identical to the corresponding position in the naturally occurring sequence. In some embodiments, a fragment is at least 5,6, 8, or 10 amino acids long. In other embodiments, the fragment is 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 comprising a fragment that has substantial identity to some reference amino acid sequence and substantially the same function or activity as the reference amino acid sequence. Typically, polypeptide analogs comprise conservative amino acid substitutions (or insertions or deletions) relative to a reference sequence. Analogs can be at least 20 or 25 amino acids long, or can be at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids long or longer, or often can be as long as a 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 as compared to the germline amino acid sequence. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art following the teachings of the present specification.

In certain embodimentsWherein the amino acid substitution of the P-cadherin antibody, or antigen-binding portion thereof, is: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered affinity for formation of protein complexes, and (4) conferring or modifying other physicochemical or functional characteristics of such analogs, while retaining specific binding to P-cadherin. Analogs can include various substitutions to the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in a portion of the polypeptide outside of the domains that form intermolecular contacts). Amino acid substitutions may also be made in domains that form intermolecular contacts that may facilitate polypeptide activity. Conservative amino acid substitutions should not significantly alter the structural properties of the parent sequence; for example, the substitution of amino acids should not alter the antiparallel beta-sheet that makes up the immunoglobulin binding domain present in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. Typically, glycine or proline is not used for antiparallel beta-sheets. Examples of polypeptide secondary and tripolar structures identified in the art are described inProteins，Structures and Molecular Principles(Creighton，Ed.，W.H.Freemanand Company，New York(1984))；Introduction to Protein Structure(c.brandennd j.tooze, eds., Garland Publishing, New York, n.y. (1991)); and Thornton, et al, Nature, 354: 105(1991), which is incorporated herein by reference.

The term "antibody" is used herein synonymously with immunoglobulin, as understood generally as known in the art. In particular, the term antibody is not limited by any particular method of producing the antibody. For example, the term antibody includes, but is not limited to, 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 antibody fragments that have the ability to specifically bind to an antigen (e.g., P-cadherin). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Is included in the termExamples of binding fragments in the "antigen-binding portion" of an antibody include (i) a Fab fragment consisting of V_L、V_H、C_LAnd C_H1 domain; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) from V_HAnd C_H1 domain; (iv) v by one-armed antibody_L and V_H(iv) an Fv fragment consisting of the domain, (V) a dAb fragment (Ward, et al., Nature, (1989) 341: 544-546) consisting of V_HDomain composition; and (vi) an isolated Complementarity Determining Region (CDR). In addition, although two domains of the Fv fragment, V_LAnd V_HAre encoded by separate genes, but they can be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain in which V is present_LAnd V_HRegion pairs form 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)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which V_HAnd V_LDomains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, et al, Proc. Natl. Acad. Sci. USA, 90: 6444-.

In addition, an antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. The use of such immunoadhesion molecules including the streptavidin core region to make tetrameric scFv molecules (Kipriyanov, et al, Human Antibodies and hybrids, 6: 93-101(1995)) and cysteine residuesUse of a base, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al, mol. Immunol., 31: 1047-one 1058 (1994)). Other examples include one or more CDRs from an antibody being covalently or non-covalently incorporated into a molecule as an immunoadhesin that specifically binds an antigen of interest (e.g., P-cadherin). In such embodiments, the CDRs may be integrated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be non-covalently integrated. Antibody moieties such as Fab and F (ab')₂Fragments can be prepared from whole antibodies using conventional techniques (e.g., papain or pepsin digestion of whole antibodies, respectively). In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

When reference is made to "antibodies" in relation to the present invention, it will be understood that antigen binding portions thereof may also be used. The antigen binding portion competes with the intact antibody for specific binding. General referenceFundamental ImmunologyCh.7(Paul, w., ed., 2nd ed. raven Press, n.y. (1989)) (incorporated by reference in its entirety for all purposes). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, the antigen binding portion includes Fab, Fab ', F (ab')₂Fd, Fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, and polypeptides comprising at least one antibody moiety sufficient to confer specific antigen binding to the polypeptide. In embodiments having one or more binding sites, the binding sites may be the same or different from each other.

The term "human antibody" is used herein to refer to any antibody in which the sequences of the variable and constant domains are human sequences. The term includes antibodies that carry sequences from human genes but have been altered to reduce potential immunogenicity, increase avidity, eliminate cysteines that may cause undesired folding, and the like. The term also includes such antibodies produced recombinantly in non-human cells, which may confer glycosylation atypical for human cells. These antibodies can be prepared in a variety of ways, as described below.

The term "chimeric antibody" as used herein refers to an antibody comprising regions from two or more different antibodies, including antibodies from different species. For example, one or more CDRs of the chimeric antibody can be from a human P-cadherin antibody. In one example, a CDR from a human antibody can bind to a CDR from a non-human antibody (e.g., mouse or rat). In another example, all CDRs can be from a human P-cadherin antibody. In another example, CDRs from more than one human P-cadherin antibody may be combined in one chimeric antibody. For example, a chimeric antibody can comprise the CDRs 1 from the first human P-cadherin antibody light chain, the CDRs 2 from the second human P-cadherin antibody light chain, and the CDRs 3 from the third human P-cadherin antibody light chain, and the CDRs of the heavy chain can be from one or more other P-cadherin antibodies. Alternatively, the framework regions may be derived from one P-cadherin antibody (from which one or more CDRs are derived) or from one or more different human antibodies. In addition, the term "chimeric antibody" is intended to include any of the above combinations, wherein the combination involves both human and non-human antibodies.

The term "humanized antibody" as used herein refers to antibodies of non-human origin in which amino acid residues characteristic of the sequence of an antibody belonging to a non-human species are replaced with residues present at corresponding positions in a human antibody. This "humanization" process is believed to reduce the immunogenicity of the produced antibody in humans. It will be appreciated that antibodies of non-human origin may be humanized using techniques well known in the art. See, e.g., Winter, et, immunol. 43-46(1993). The antibody of interest can be processed by recombinant DNA techniques to replace CH1, CH2, CH3, the hinge domain and/or the framework domain with the corresponding human sequence. See, for example, WO92/02190 and U.S. patent nos.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 within its meaning chimeric human antibodies and CDR-grafted antibodies. Chimeric human antibodies of the invention include V of non-human species antibodies_HAnd V_LAnd C of human antibody_HAnd C_LA domain. CDR-grafted antibodies of the invention are prepared by grafting human antibodies V_HAnd V_LRespectively with V of a non-human animal antibody_HAnd V_LInstead, it is obtained.

The term "ELISA" is used herein to refer to enzyme-linked immunosorbent assays. Such assays are well known to those skilled in the art. Examples of such assays can be found in Vaughan, t.j., et al, nat.biotech, 14: 309, 314(1996) and example 7 of the present application.

The term "surface plasmon resonance" as used herein refers to the use of BIACORE, for example^TMThe system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.) allows the analysis of the optical phenomenon of real-time biospecific interactions by detecting changes in protein concentration in the Biosensor matrix. See Jonsson et al, ann.biol.clin., 51: 19-26 (1993); jonsson, et al, Biotechniques, 11: 620-627 (1991); jonsson, et al, j.mol.recognit, 8: 125-131 (1995); and Johnsson, et al, anal. biochem., 198: 268-277(1991).

The term "K_D"refers to the avidity equilibrium constant for a particular antibody-antigen interaction. When K is_DAn antibody is said to specifically bind to an antigen at less than or equal to 1mM, preferably less than or equal to 100nM and most preferably less than or equal to 10 nM. K_DThe affinity constant can be measured by surface plasmon resonance, e.g., using BIACORE as described in example 6^TMAnd (4) preparing the system.

The term "k_off"refers to the dissociation rate constant for a particular antibody-antigen interaction. k is a radical of_offThe dissociation rate constant can be measured by surface plasmon resonance, for example using BIACORE as described in example 6^TMAnd (4) preparing the system.

The term "P-cadherin-dependent cell adhesion assay" as used herein refers to an assay for measuring P-cadherin resistanceThe body blocks the ability of cells to adhere to the receptor P-cadherin which is already immobilized on a solid support. This type of assay can be performed, for example, by immobilizing P-cadherin on a solid support (e.g., plastic). Cells overexpressing P-cadherin are then allowed to adhere to the solid support via P-cadherin-P-cadherin interactions. The level of adhesion can be quantified with or without the use of P-cadherin antibodies. Adhesion as a function of antibody concentration can then be used to determine IC₅₀The value is obtained. Example 3 provides methods for measuring P-cadherin antibody IC₅₀Additional details of the P-cadherin-dependent cell adhesion assay of values.

The term "P-cadherin-dependent cell aggregation assay" as used herein refers to an assay for measuring the ability of a P-cadherin antibody to block the aggregation of cells expressing P-cadherin on their surface. For example, this type of assay may use a cell line that overexpresses P-cadherin, where the cells are placed in suspension and allowed to form P-cadherin-dependent aggregates. The ability of the P-cadherin antibody to prevent this aggregation is then quantified using an aggregation assay by measuring the size of cell aggregates obtained with and without the antibody. The size of cell aggregates as a function of P-cadherin antibody concentration can then be used to determine IC₅₀The value is obtained. Example 4 provides methods for measuring several P-cadherin antibody ICs₅₀Additional details of the P-cadherin-dependent aggregation assay of values.

The term "P-cadherin-dependent spheroid disruption assay" as used herein refers to an assay for measuring the ability of P-cadherin antibodies to disrupt preformed P-cadherin-dependent cell aggregates. By measuring aggregate size reduction as a function of antibody concentration, IC can be determined₅₀The value is obtained. Example 5 provides methods for measuring P-cadherin antibody IC₅₀Additional details of the P-cadherin-dependent spheroid disruption assay of values.

The term "molecular selectivity" is used herein to refer to the affinity of an antibody for a particular antigen being greater than its affinity for the relevant antigen. For example, an antibody of the invention is selected for P-cadherin relative to E-cadherin, i.e., the antibody has at least 2-fold, e.g., 4-fold, or 10-fold, or 50-fold, or 100-fold or more, greater affinity for P-cadherin than for E-cadherin. Such affinity can be measured using standard techniques known to those skilled in the art.

The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface clusters of molecules (e.g., amino acids or carbohydrates or sugar side chains) and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all the interaction points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In conformational epitopes, the interaction sites are present across amino acid residues on the protein that are separated from each other. Once the epitope of interest on an antigen is identified, it is possible to generate antibodies against that epitope using, for example, the techniques described herein. Alternatively, in the discovery process, the production and characterization of antibodies may set forth information about the epitope of interest. From this information it is possible to subsequently competitively screen antibodies for binding to the same epitope. One way to achieve this is to construct a cross-competition study to find antibodies that compete for binding to each other, i.e., the antibodies compete for binding to the antigen. A high-throughput method for "binding" antibodies based on their cross-competition is described in International patent publication No. WO03/48731.

The term "competes" is used herein with respect to an antibody to refer to a first antibody or antigen-binding portion thereof that competes for binding with a second antibody or antigen-binding portion thereof, wherein binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody as compared to binding of the first antibody in the absence of the second antibody. Alternatively, it may be (but is not necessarily) the case that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody is capable of inhibiting the binding of the second antibody to its epitope, while the second antibody is not capable of inhibiting the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of another antibody to its epitope or ligand, to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitope. Both competing and cross-competing antibodies are included in the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof, etc.), the skilled artisan will appreciate that such competing and/or cross-competing antibodies are included and suitable for use in the methods disclosed herein in light of the teachings provided herein.

The term "utilized" with respect to a particular gene is used herein to mean that the amino acid sequence of a particular region in an antibody is primarily derived from the gene as the B-cell matures. For example, the phrase "utilizing human V_H-the heavy chain variable region amino acid sequence of a family 3 gene "refers to the following substitutions: v of antibody at B-cell maturation_HThe region is from a gene fragment of the VH-3 family. There are more than 30 different functional heavy chain variable genes in human B cells, which are used to produce antibodies. Thus, the use of specific heavy chain variable genes indicates preferred binding motifs for antibody-antigen interactions, in terms of combined characteristics of binding to antigen and functional activity. It is recognized that gene utilization analysis provides only a limited structural profile of antibodies. Because human B-cells randomly produce V-D-J heavy chain or V-J kappa light chain transcripts, there are a number of secondary processes including, but not limited to, somatic hypermutation, n-addition, and CDR3 expansion. See, e.g., Mendez et al Nature Genetics 15: 146-156(1997).

Twenty conventional amino acids and their abbreviations are used herein according to conventional usage. See alsoImmunology-A Synthesis(2^ndEdition, e.s. golub and d.r.gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

The term "polynucleotide" as referred to herein refers to a polymeric form of nucleotides of at least 10 bases in length, which are either ribonucleotides or deoxynucleotides or modified forms of both types of nucleotides. The term includes single-stranded and double-stranded forms.

The term "isolated polynucleotide" is used herein to refer to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, as the source of the "isolated polynucleotide" is (1) not accompanied by all or part of the polynucleotide as the "isolated polynucleotide" is naturally found, (2) operably linked to a polynucleotide to which the polynucleotide is not naturally linked, or (3) not naturally occurring as part of a larger sequence.

The term "naturally occurring nucleotide" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkage" as referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroamidate, and the like. See, e.g., LaPlanche, et al, nucleic acids res, 14: 9081 (1986); stec, et al, j.am.chem.soc., 106: 6077 (1984); stein, et al, nucleic acids res, 16: 3209 (1988); zon, et al, Anti-Cancer drug design, 6: 539 (1991); zon, et al.,Oligonucleotides and Analogues：A Practical Approachpp.87-108(f. eckstein, ed., Oxford University Press, Oxford england (1991)); U.S. patent nos.5,151,510; uhlmann and Peyman, chemical reviews, 90: 543(1990), the disclosure of which is incorporated herein by reference. The oligonucleotide may include a label for detection, if desired.

"operably linked" sequences include expression control sequences that are adjacent to the gene of interest and expression control sequences that act in reverse or remotely to control the gene of interest.

The term "expression control sequence" as used herein refers to polynucleotide sequences necessary to effect the expression and processing of the coding sequences to which they are ligated. Expression control 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 (e.g., Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance protein secretion when desired. The nature of such regulatory sequences varies depending on the host organism; in prokaryotes, such regulatory sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such regulatory sequences typically include promoters and transcription termination sequences. The term "control sequences" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

The term "vector" as used herein refers to a nucleic acid molecule capable of transferring another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA portion into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, a vector (e.g., a non-episomal mammalian vector) can be integrated into the genome of a host cell by introduction into the host cell, and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably 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 refers to a cell into which a recombinant expression vector has been introduced. It is understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "germline" is used herein to refer to the nucleotide sequences of antibody genes and gene fragments as they are passed from parent to offspring by germ cells. This germline sequence is distinct from nucleotides encoding antibodies in mature B cells that have been altered by recombination and hypermutation events during B cell maturation.

The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues in the two sequences that are identical when aligned for maximum correspondence. The length of the sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least 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. There are a number of different algorithms available in the art for measuring nucleotide sequence identity. For example, the polynucleotide sequences can be compared using FASTA, Gap or Bestfit (which is a program in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin). FASTA, including programs such as FASTA2 and FASTA3, provides alignments and percentage sequence identity of regions of optimal overlap between 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); incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, the percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size 6, NOPAM factor for the integration matrix) or using Gap with the default parameters provided in GCG Version 6.1, incorporated herein by reference.

Unless otherwise indicated, reference to a nucleotide sequence includes its complement. Thus, the meaning of a nucleic acid having a particular sequence should be understood to include the complementary strand thereof and the complementary sequence thereof.

The term "significant similarity" or "significant sequence similarity", when referring to a nucleic acid or fragment thereof, refers to the optimal alignment with another nucleic acid (or its complementary strand) for appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm (e.g., FASTA, BLAST, or Gap, as described above): nucleotide sequence identity exists in 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.

The term "percent sequence identity" in the context of amino acids refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison may be over 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. There are a number of different algorithms 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 Bestfit (which is a program in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin).

The term "significant identity" or "significant similarity", as applied to polypeptides, means that two amino acid sequences share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, more preferably at least 97%, 98% or 99% sequence identity, when optimally aligned, for example by the programs GAP or BESTFIT using default GAP weights provided by the programs. In certain embodiments, the different residue positions differ by conservative amino acid substitutions.

"conservative amino acid substitutions" are used herein to refer to the following amino acid substitutions: wherein one amino acid residue is substituted with another amino acid residue bearing a side chain R group with similar chemical characteristics (e.g., charge or hydrophobicity). In general, conserved amino acid residues do not significantly alter the functional characteristics of proteins. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, Methods mol. biol., 243: 307-31(1994). Examples of amino acid groups having side chains with similar chemical characteristics include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) an aromatic side chain; phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. For example, a conservative amino acid substitution group may be: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

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

Sequence identity of polypeptides is typically measured using sequence analysis software. Protein analysis software uses similarity measures that assign values to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match sequences. For example, GCG contains software such as "Gap" and "Bestfit" that can use default parameters specified by the program to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different species of organisms) or between wild-type proteins and their analogs. See, for example, GCG Version 6.1(University of Wisconsin, WI). Polypeptide sequences can also be compared using FASTA with default or recommended parameters, see GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentage sequence identity of the best overlapping regions between query and search sequences (Pearson, Methods enzymol., 183: 63-98 (1990); Pearson, Methods mol. biol., 132: 185-219 (2000)). Another preferred algorithm when comparing the sequences 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 provided with the program. See, e.g., Altschul, et al, j.mol.biol., 215: 403-; altschul, et al, Nucleic Acids res, 25: 3389-402(1997).

In general, the length of polypeptide sequences for homology comparison will 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 in a database comprising a large number of sequences from different organisms, it is preferred to compare the amino acid sequences.

The term "tag" or "labeled" is used herein to refer to an antibody that has incorporated into it another molecule. In one embodiment, the tag is a detectable label, e.g., incorporating a radiolabeled amino acid, or a biotin-based moiety is attached to the polypeptide, which is detectable by labeled avidin (e.g., streptavidin containing a fluorescent label, or enzyme activity detectable by optical or colorimetric methods). In another embodiment, the label or marker may be therapeutic, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Tags for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Fluorescent tags (e.g., FITC, rhodamine, lanthanide phosphors), enzyme tags (e.g., horseradish peroxidase,. beta. -galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, polypeptide epitopes recognized by secondary reporters that are predetermined (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, epipodophyllotoxin (etoposide), podophyllotoxin (tenoposide), vincristine (vincristine), vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthradione (dihydroanthraquinone), mitoxantrone hydrochloride (mitoxantrone), mithramycin, rhodanamycin, beta-galactosidase, luciferase, alkaline phosphatase, and the like, Actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogues or homologues thereof. In some embodiments, the tags are attached by spacer arms of various lengths to reduce potential steric hindrance.

By "therapeutically effective amount" is meant an amount of the therapeutic agent administered that will alleviate, to some extent, one or more of the symptoms of the disease being treated. For cancer treatment, a therapeutically effective amount refers to an amount that has at least one of the following effects: reducing the size of the tumor; inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; inhibit (i.e., slow to some extent, preferably stop) tumor growth to some extent and alleviate (or preferably eliminate) to some extent one or more symptoms associated with cancer.

"treating" or "treatment" refers to the reduction or elimination of a biological disorder and/or an accompanying symptom. In the case of cancer, these terms simply mean that the life expectancy of the individual affected by the cancer will be extended, or one or more symptoms of the disease will be alleviated.

"contacting" refers to the association of an antibody of the invention, or an antigen-binding portion thereof, with a target P-cadherin protein, or a surface thereof, in such a way that the antibody is capable of affecting the biological activity of the P-cadherin proteinThe bits are grouped together. Such "contacting" may be done "in vitro", i.e., in a test tube, petri dish, etc. In vitro, the contacting may involve only the antibody or antigen binding portion thereof and the P-cadherin protein or epitope thereof, or it may involve the entire cell. The cells may be maintained or cultured in a cell culture dish and contacted with the antibody or antigen-binding portion thereof in this environment. In this context, the ability of a particular antibody, or antigen-binding portion thereof, to affect a P-cadherin-associated disease (i.e., the IC of the antibody) can be determined prior to attempting use of the antibody in more complex in vivo assays₅₀). For cells outside the biological body, various methods of contacting the P-cadherin with an antibody or antigen-binding portion thereof exist and are well known to those skilled in the art.

Unless otherwise indicated, "abnormal cell growth" as used herein refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition), including abnormal growth of normal cells and growth of abnormal cells. This includes, but is not limited to, abnormal growth of: tumor cells (tumors) that proliferate by expressing a mutated tyrosine kinase or overexpressing a receptor tyrosine kinase; benign or malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs; any tumor that proliferates through receptor tyrosine kinases; any tumor that proliferates through aberrant serine/threonine kinase activation; benign and malignant cells of other proliferative diseases in which abnormal serine/threonine kinase activation occurs; benign and malignant tumors expressing the activated Ras oncogene; benign or malignant tumor cells, which Ras protein as another gene in oncogenic mutation results in activation; other proliferative diseases of benign or malignant cells, in which abnormal Ras activation occurs. Examples of such benign proliferative diseases are psoriasis, benign prostatic hypertrophy, Human Papilloma Virus (HPV) and restinosis. "abnormal cell growth" also relates to and includes abnormal growth of benign and malignant cells caused by the enzymatic activity of farnesyl protein transferase.

The terms "abnormal cell growth" and "hyperproliferative disease" are used interchangeably in this application.

"in vitro" refers to a step that is performed in an artificial environment (such as, but not limited to, in a test tube or culture medium).

By "in vivo" is meant a procedure performed in vivo (such as, but not limited to, a mouse, rat, or rabbit).

Brief description of the drawings

FIG. 1 shows SEQ ID NOs: 1-347, in a nucleic acid sequence.

Detailed Description

Human P-cadherin antibodies

The present invention relates to isolated human antibodies, or antigen-binding portions thereof, that bind to human P-cadherin. Preferably, the human antibody is a recombinant human P-cadherin antibody having greater affinity for P-cadherin than for E-cadherin. Aspects of the invention relate to such antibodies and antigen-binding portions and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such antibodies and antigen-binding portions. Methods of using the antibodies and antigen-binding portions of the invention to detect human P-cadherin or to inhibit the activity of human P-cadherin in vivo or in vitro are also encompassed by the invention.

P-cadherin amino acid and nucleotide sequences from several species, including humans, are known (see, e.g., accession No. nm — 001793.3). Human P-cadherin or antigenic portions thereof can be prepared according to 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, an antibody of the invention is an antibody such as 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; 200-h 06; g-194-b 09; g-194-g 09; g-196-g 03; g-196-h 02; g-194-e 01; g-194-e 06; 129-1c 4; and g-12IgG denoted by 9-1c 4. As discussed in more detail in example 1, high throughput screening using scFv phage display libraries was used to identify 129-1c4 scFv, which was subsequently converted to IgG. 129-1c4 represents the lead antibody identified in the primary phage display screen and is the parent antibody of one lineage from which several other antibodies of the invention were derived. Several such derivatized antibodies are designated 194-e 06; 194-a 02; 194-b 09; 195-e 11; 194-g 09; 196-h 02; 194-e 01; 196-d 10; 196-g 03; 196-e 06; 195-a 09; 198-a 09; and 200-h06, and represents an optimized antibody in the 129-1c4 parental lineage. The g-129-1c4 antibody is a germline version of the 129-1c4 parent antibody, wherein V is_HAnd V_LCertain amino acids in the framework regions of the domains are mutated to match amino acids in the germline framework regions. Any of the antibodies described above may also be germlined such that the framework region sequences are identical to the germline framework sequences in g-129-1c 4. For example, in one embodiment of the invention, antibodies g-194-b09, g-194-g09, g-196-g03, g-196-h02, g-194-e01, g-195-e11, g-200-h06, and g-194-e06 are germlined versions of 194-b09, 194-g09, 196-g03, 196-h02, 194-e01, 195-e11, 200-h06, and 194-e06, respectively. It will be apparent to those skilled in the art that the particular amino acid that is mutated to achieve a germlined version can be obtained by comparing a germlined antibody to an unlistened antibody. As discussed below, specific amino acid sequences of the antibodies of the invention are depicted in tables 1-3 and fig. 1.

There is a strong tendency to utilize V of the heavy chain variable region_H3 Gene family the antibodies of the invention are produced. In particular, the 129-1c4 parent antibody is derived from V_H3-23 variable gene fragment. In human B cells, there are more than 30 different functional heavy chain variable genes that are used to produce antibodies. However, the combined features of antigen binding and functional activity tend to be predictive of preferred binding motifs for antibody-antigen interactions.

It is recognized that gene utilization assays provide only a limited structural profile of antibodies. Because human B-cells randomly produce V-D-J heavy chain or V-J kappa light chain transcripts, there are a number of secondary processes including, but not limited to, somatic hypermutation, n-addition, and CDR3 expansion. See, e.g., Mendez et al Nature Genetics 15: 146-. Therefore, in order to further examine the structure of the antibody of the present invention, an antibody of a predetermined amino acid sequence was produced from a cDNA obtained from a clone. In addition, the N-terminal amino acid sequence was obtained by protein sequencing. FIG. 1 provides the nucleotide and amino acid sequences of several heavy and light chain variable regions of the antibodies of the invention.

As shown in tables 1 and 2, each of the specific antibodies described above can pass through their heavy chain (V)_H) And light chain (V)_L) The variable domain sequence of (a). The specific sequences involved in these SEQ ID nos. are shown in fig. 1. As shown in tables 1 and 2, V corresponding to the above antibody_HAnd V_LThe amino acid and DNA sequences of SEQ ID NOs: 1-23, 68-90, and 320-.

TABLE 1

Human P-cadherin antibodies
					Antibodies	Sequence identifier (SEQ ID NO:)
VH		VL
				Amino acids		DNA	Amino acids	DNA
194-e06	1	68	14						81
				194-a02	2	69	14	81
194-b09	2	69	15						82
				195-e11	3	70	16	83
194-g09	4	71	17						84
				196-h02	4	71	23	90
194-e01	5	72	18						85
				196-d10	6	73	23	90
196-g03	7	74	23						90
				196-e06	8	75	23	90
195-a09	9	76	23						90

Human P-cadherin antibodies
					Antibodies	Sequence identifier (SEQ ID NO:)
VH		VL
				Amino acids		DNA	Amino acids	DNA
198-a09	10	77	19						86
				200-h06	11	78	20	87
129-1c4	12	79	21						88

TABLE 2

Germlined human P-cadherin antibodies
					Antibodies	Sequence identifier (SEQ ID NO:)
VH		VL
				Amino acids		DNA	Amino acids	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-e01	324	336	330						342
				g-194-e06	325	337	331	343
g-129-1c4	13	80	22						89

Additional antibodies and antigen-binding portions of the invention can also be described as comprising CDR and FR sequences that make up the heavy and light chain variable regions of the antibodies shown in tables 1 and 2. Thus, SEQ ID nos. corresponding to various CDR and FR sequences of the antibodies of the invention are shown in table 3. In addition, a number of random mutations were also made in the heavy and light chain CDR3 regions of the 129-1c4 parent antibody, which resulted in an improved affinity for P-cadherin, as measured by an epitope competition assay, in the range of 10-to 417-fold improvement (see example 8). These mutated V_HAnd V_LThe SEQ ID NOs of the CDR3 sequence (SEQ ID NOs: 91-256 and 257-319) are also included in Table 3 below.

TABLE 3

SEQ ID NOs：	Description of the invention
		24	VH CDR1
25	VH CDR2
		26-37，91-256	VH CDR3
38	VL CDR1
		39	VL CDR2
40-47，257-319	VL CDR3
		48	VH FR1
49	VH FR2
		50-55	VH FR3
56，57	VH FR4
		58，59	VL FR1
60-62	VL FR2
		63-66	VL FR3
67	VL FR4

Method for producing antibody

Phage display libraries

The antibodies or antigen binding portions of the invention can be prepared according to several methods known in the art. For example, phage display technology can be used to provide libraries containing a series of antibodies with different affinities for P-cadherin. These libraries can then be screened to identify and isolate isolated antibodies having the desired affinity for P-cadherin.

For example, recombinant human P-cadherin antibodies of the invention can be isolated by screening a library of recombinant combinatorial antibodies. Preferably, the library is human V_LAnd V_HA scFv phage display library produced from cDNA prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Kits for producing true libraries of bacteriophages are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01; and the Stratagene SurfZAP^TMPhage display kit, catalog No. 240612). There are also other methods and reagents available for generating and screening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT publication Nos. WO92/18619, WO91/17271, WO92/20791, WO92/15679, WO93/01288, WO92/01047 and WO 92/09690; Fuchs et al, Bio/Technology 9: 1370-1372 (1991); Hay, et al, hum.Antibod.Hybridomas, 3: 81-85 (1992); Huse, et al, Science, 246: 1275-Bu 1281 (1989); McCafferty et al, Nature, 348: 552-554 (1990); Grifts, et al, EMBO et al., EMBOJ, 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 al, nuc.acid res, 19: 4133 4137 (1991); barbas, et al, proc.natl.acad.sci.usa, 88: 7978-7982 (1991); and Griffiths, et al, emboj., 13: 3245-3260 (1994); all incorporated herein by reference).

Another method for preparing antibody libraries for use in phage display technology comprises the steps of: immunizing a non-human animal comprising a human immunoglobulin locus with P-cadherin or an antigenic portion thereof to generate an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding the heavy and light chains of the antibody of the invention from the extracted cells; reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers and inserting the cDNA into a phage display vector such that the antibody is expressed on the phage. In order to produce such a series, it is not necessary to immortalize B cells from the immunized animal. Alternatively, naive B cells can be used directly as a source of DNA. cDNA mixtures obtained from B cells (e.g., from the spleen) are used to prepare expression libraries, e.g., phage display libraries transfected into e. Fundamentally, clones from the library that produce the desired strength affinity for the antigen are identified, and the DNA encoding the product responsible for such binding is recovered and processed for standard recombinant expression. Phage display libraries can also be constructed using previously processed nucleotide sequences and screened in a similar manner. Typically, cdnas encoding the heavy and light chains are provided independently or linked to form Fv analogs for production in phage libraries. The phage library is then screened for antibodies with the highest affinity for P-cadherin, and genetic material is recovered from the appropriate clones. Further rounds of screening can improve the affinity of the original antibody isolated.

In one embodiment, to isolate and produce human P-cadherin antibodies with desired properties, the human P-cadherin antibodies described herein are first used to select human heavy and light chain sequences having similar binding activity to P-cadherin using the epitope imprinting method described in PCT publication No. wo93/06213, which is incorporated herein by reference. Preferably, the antibody library used in the method is a library as described in PCT publication No. wo92/01047, McCafferty, et al, Nature, 348: 552 (1990); and Griffiths, et al, EMBO j.12: 725, 734(1993) the scFv libraries prepared and screened, the data of which are incorporated herein by reference. The scFv antibody library can be screened using human P-cadherin as an antigen. The phage library was screened for antibodies with the highest affinity for P-cadherin, and genetic material was recovered from the appropriate clones. Further rounds of screening can improve the affinity of the original antibody isolated.

Once the initial person V is selected_LAnd V_HDomains, can then be subjected to a "mix and match" experiment in which the initially selected different pairs of V are screened_LAnd V_HBinding of fragments to P-cadherin to select preferred V_L/V_HAnd (4) combining. These mixing and matching experiments can also be performed on random mutations V_LAnd V_HFragmentation is followed for optimal binding as described below. In addition, V is preferable for further improvement of antibody quality_L/V_HPair of V_LAnd V_HFragments may be randomly mutated (preferably at V) in the following manner_HAnd/or V_LIn the CDR3 region) similar to the in vivo somatic mutation approach responsible for antibody affinity maturation in the innate immune response. This in vitro affinity maturation can be achieved, for example, by using a peptide that binds to V separately_HCDR3 or V_LPCR primer amplification V with complementary CDR3_HOr V_LDomains, the primers having been "punctured" (spiked) with a random mixture of four nucleotide bases at certain positions, such that the resulting PCR product encodes the V described below_HAnd V_LFragment of the V_HAnd V_LRandom mutations in the fragments were introduced into V_HAnd/or V_LCDR3 region. V which can be mutated at these random_HAnd V_LIn the fragmentScreening for binding to P-cadherin, sequences exhibiting high affinity and low off-rate (off-rate) for P-cadherin can be selected. As previously described, SEQ ID NOs: 91-256 and 257-319 indicate that several V's of the invention, which are randomly mutated and show improved affinity, are present_HAnd V_LCDR3 sequence.

After screening and isolating the P-cadherin antibodies of the invention from the recombinant immunoglobulin display library, the nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid may be further processed to produce other antibody formats of the invention, as described below. To express recombinant human antibodies isolated by screening combinatorial libraries, the DNA encoding the antibodies is cloned into a recombinant expression vector and introduced into host cells, as described below.

Immunization

In another embodiment, human P-cadherin antibodies can be produced by immunizing a non-human, transgenic animal whose genome comprises some or all of the human immunoglobulin heavy and light chain loci that carry the P-cadherin antigen. For example, the non-human animal can be a XeNOMOUS^TMAn animal. (Abgenix, Inc., Fremont, CA).

XENOMOUSE^TMMice were manipulated into mouse strains that contain large-fragment human immunoglobulin heavy and light chain loci that are deficient in mouse antibody production. See, e.g., Green, et al, nature genetics, 7: 13-21(1994) and U.S. patent nos.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 WO91/10741, WO94/02602, WO96/34096, WO96/33735, WO98/16654, WO98/24893, WO98/50433, WO99/45031, WO99/53049, WO00/09560 and WO 00/037504.

The methods disclosed in these documents can be modified as described in U.S. patent No.5,994,619, which is incorporated herein by reference. U.S. patent No.5,994,619 describes a method for making newly Cultured Inner Cell Mass (CICM) cells and cell lines (from swine and cattle), and transgenic CICM cells into which heterologous DNA is inserted. CICM transgenic cells can be used to make cloned transgenic embryos, fetuses, and progeny. The' 619 patent also describes methods for making transgenic animals capable of delivering heterologous DNA to their offspring. Examples of non-human animals that can be used in these methods include rats, sheep, pigs, goats, cattle, chickens, and horses.

XENOMOUSE^TMMice produce a fully human antibody of the human-like human series and produce antigen-specific human antibodies. In some embodiments, XENOMOUSE^TMMice contain about 80% of the human antibody V gene series by introducing measured megabases (megabases sized), germline configured fragments of the human heavy chain locus, and the kappa light chain locus in Yeast Artificial Chromosomes (YACs). In another embodiment, XENOMOUSE^TMMice also contain about all human lambda light chain loci. See Mendez, et al, Nature Genetics, 15: 146-: 483-495(1998) and WO98/24893, the disclosures of which are incorporated herein by reference.

In some embodiments, the non-human animal comprising human immunoglobulin genes is an animal having a human immunoglobulin "minilocus" (minilocus). In the small locus pathway, the foreign Ig locus is mimicked by the inclusion of individual genes from the Ig locus. Thus, one or more V_HGene, one or more D_HGene, J or J_HThe gene, the mu constant domain and the further constant domain (preferably the gamma constant domain) form one construct for insertion into an animal. This pathway is described in U.S. patent nos.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.

The non-human animal can then be immunized with the P-cadherin antigen as described below under conditions that allow for antibody production. Isolating antibody-producing cells from the animal, isolating nucleic acids encoding the heavy and light chains of the P-cadherin antibody of interest from the isolated antibody-producing cells or from an immortalized cell line produced by such cells. These nucleic acids are then processed using techniques known to those skilled in the art and as described further below to reduce the amount of non-human sequences, i.e., to humanize the antibody to reduce the immune response in humans.

In some embodiments, the P-cadherin antigen may be isolated and/or purified P-cadherin. In some embodiments, the P-cadherin is human P-cadherin. In other embodiments, the P-cadherin antigen can be a cell that expresses or overexpresses P-cadherin. In other embodiments, the P-cadherin antigen is a recombinant protein expressed by recombinant technology from yeast, insect cells, bacteria such as e. Immunization of animals can be carried out by any method known in the art. See for example Harlow and Lane,Antibodies：A Laboratory Manualnew York: 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, for example, Harlow and Lane, supra, and U.S. patent No.5,994,619. For example, the P-cadherin antigen may be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immune stimulating complex). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a localized deposit, or they may comprise substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system (chemotactics). Preferably, if the polypeptide is administered, the immunization schedule will involve two or more administrations of the polypeptide (over several weeks).

For example, following immunization of a transgenic animal with P-cadherin as described above, primary cells (e.g., spleen or peripheral blood B cells) can be isolated from the immunized transgenic animal, and individual cells producing antibodies specific for the antigen of interest can be identified. The polyadenylated mRNA from each individual cell is then isolated and reverse transcription polymerase chain reaction (RT-PCR) is performed using sense primers that anneal to the variable region sequences (e.g., degenerate primers that recognize the FR1 region of most or all of the human heavy and light chain variable region genes, and antisense primers that anneal to the constant region or linker sequences). The cdnas for the heavy and light chain variable regions are then cloned and expressed in any suitable host cell (e.g., myeloma cells) as chimeric antibodies with respective immunoglobulin constant regions, e.g., heavy chain and kappa or lambda constant domains. See Babcook, et al, proc.natl.acad.sci.usa, 93: 7843-48, (1996), incorporated herein by reference. P-cadherin can then be identified and isolated as described herein.

Recombinant method for producing antibodies

The antibodies or antibody portions 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 recombinantly, a host cell is transfected with one or more recombinant expression vectors with DNA segments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell, preferably secreted into the culture medium in which the host cell is cultured, from which medium the antibody can be recovered. Antibody heavy and light chain genes are obtained using standard recombinant DNA methods, such as Sambrook, Fritsch and Maniatis (eds), incorporating these genes into recombinant expression vectors and introducing the vectors into host cells,Molecular Cloning；A Laboratory Manual，Second Edition，Cold Spring Harbor，N.Y.，(1989)，Ausubel，F.M.，et al.(eds.)Current Protocols in Molecular Biologythe disclosures of these documents are incorporated herein by reference, as described in Greene Publishing Associates, (1989) and U.S. Pat. No.4,816,397.

Mutations and modifications

To express the P-cadherin antibodies of the invention, the encoding V may first be obtained using any of the methods described above_HAnd V_LDNA fragments of the regions. Various mutations, deletions and/or additions may also be introduced into the DNA sequence using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods (e.g., PCR-mediated mutagenesis) in which mutated nucleotides are incorporated into PCR primers such that the PCR product contains the desired mutation or site-directed mutagenesis. For example, one type of substitution that can be made is to alter one or more cysteines in an antibody that can be chemically reactive to another residue (such as, but not limited to, alanine or serine). For example, substitutions of non-canonical cysteines may be present. The substitutions may be made in the CDRs or in the variable domain framework regions or in the constant domain of the antibody. In some embodiments, the cysteine is canonical.

Antibodies may also be mutated in the heavy and/or light chain variable domains, for example to alter the binding characteristics of the antibody. For example, mutations can occur in one or more CDR regions to increase or decrease the K of an antibody to P-cadherin_DIncreasing or decreasing k_offOr altering the binding specificity of the antibody. Site-directed mutagenesis techniques are well known in the art. See, e.g., Sambrook, et al and Ausubel, et al, supra, which are incorporated herein by reference. For example, as described in more detail in example 8, a number of variable V's of the 129-1c4 parent were made according to the procedure described above_HAnd V_LCDR3 sequence and is shown in fig. 1 as SEQ ID NOs: 91 to 256 (V)_HCDR3 variant) and SEQ ID NOs: 257 to 319 (V)_LCDR3 variant).

Mutations can also be made in the framework regions or constant domains to increase the half-life of the P-cadherin antibody. See, e.g., PCT publication No. WO00/09560, incorporated herein by reference. Mutations in the framework or constant domains can also be made to alter the immunogenicity of the antibody, provide sites for covalent or non-covalent binding to another molecule, or alter characteristics such as complement binding, FcR binding, and antibody-dependent cell-mediated cytotoxicity. According to the invention, a single antibody may have mutations in any one or more of the CDR or variable domain framework or constant regions.

In a process known as "germlining", V_HAnd V_LCertain amino acids in the sequence may be mutated to match in germline V_HAnd V_LNaturally occurring amino acids in the sequence. In particular, V_HAnd V_LThe amino acid sequence of the framework regions in the sequence may be mutated to match the germline sequence to reduce the risk of immunogenicity when the antibody is administered. Human V_HAnd V_LGermline DNA sequences of genes are known in the art (see, e.g., the "Vbase" human germline sequence database; see also Kabat, E.A., et al (1991))Sequences of Proteins of Immunological Interest，Fifth EditionDepartment of Health and Human Services, NIH Publication No. 91-3242; tomlinson, et al (1992) j.mol.biol.227: 776-798; and Cox, et al eur.j.immunol.24: 827-836 (1994); the contents of each material are expressly incorporated herein by reference).

Another type of amino acid substitution that can be made is the removal of possible proteolytic sites in the antibody. Such sites may be present in the CDRs or in the framework regions of variable domains or in the constant domains of antibodies. Substitution of cysteine residues and removal of proteolytic sites can reduce the risk of heterogeneity in the antibody product from 7 to improve its homogeneity. Another type of amino acid substitution is elimination of asparagine-glycine pairs by changing one or both residues, which form a possible deamidation site. In another example, the C-terminal lysine of the heavy chain of the P-cadherin antibody of the present invention can be cleaved. In various embodiments of the invention, the heavy and light chains of the P-cadherin antibody may optionally include N-terminal signal sequences, such as those set forth in SEQ ID NOs: 346 and 347, respectively.

Invention V once the code is obtained_HAnd V_LDNA fragment of fragmentThese DNA fragments can be further processed by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes, or to scFv genes. In these processes, V_L-or V_HThe encoding DNA segment is operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker (flexible linker). The term "operably linked" is used in this context to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments are maintained in-frame.

Separated code V_HDNA of the region can be obtained by converting V_HThe coding DNA is operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3) and converted into the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat, E.A., et al (1991))Sequences of Proteins of Immunological Interest，Fifth Ed., U.S. department of Health and Human Services, NIH Publication No.91-3242), DNA fragments containing these regions were obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG2 constant region. The IgG1 constant region sequence may be any allele or allotype known to exist between different individuals, such as Gm (1), Gm (2), Gm (3), and Gm (17). These allotypes represent the naturally occurring amino acid substitutions in the constant region of IgG 1. For example, the heavy chain IgG1 constant region can be SEQ ID NO: 344. with respect to the heavy chain gene of the Fab fragment, V_HThe coding DNA may be operably linked to another DNA molecule encoding only the constant region of heavy chain CH 1. The CH1 heavy chain constant region may be from any heavy chain gene.

Separated code V_LDNA of the region can be obtained by ligating V_LCoding DNA and coding light chain constant region C_LIs operably linked to be converted into a full-length light chain gene (e.g., a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat, e.a.,et al.(1991)Sequences of Proteins of Immunological Interestfifth Ed., U.S. department of Health and Human Services, NIH Publication No.91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region may be any of a variety of alleles known to occur between individuals, such as Inv (1), Inv (2), and Inv (3). The lambda constant region can be from any of the three lambda genes. For example, the light chain IgG1 constant region can be SEQ ID NO: 347.

to generate the scFv genes, V_H-and V_LEncoding DNA fragments with encoding flexible linkers (e.g.encoding amino acid sequence (G1 y)₄-Ser)₃) Is operably linked such that V_HAnd V_LThe sequence may be expressed as a single-chain protein in close proximity, where V_HAnd V_LThe regions are connected by flexible linkers (see, e.g., Bird et al science 242: 423-. Single chain antibodies may be monovalent (if only a single V is used)_HAnd V_L) Bivalent (if two V's are used)_HAnd V_L) Or multivalent (if more than two V's are used)_HAnd V_L). Bispecific or multivalent antibodies can be generated that specifically bind to P-cadherin and another molecule.

In another embodiment, a fusion antibody or immunoadhesin can be made comprising all or part of a P-cadherin antibody of the invention linked to another polypeptide. In another embodiment, only the variable region of the P-cadherin antibody is linked to the polypeptide. In another embodiment, V of the P-cadherin antibody_HThe domains are linked to the first polypeptide, and the V of the P-cadherin antibody_LThe domain is linked to a second polypeptide which is linked to the first polypeptide in the following manner: v_HAnd V_LThe domains are capable of interacting with each other to form an antigen binding site. In another preferred embodiment, V_HDomains with V_LThe domains are separated by a linker, such that V_HAnd V_LThe domains may interact with each other. Then V is put_H-linker-V_LThe antibody is linked to a polypeptide of interest. In addition, fusion proteins can be produced in which two (or more) single-chain antibodies are linked to each other. This applies when it is desired to produce bivalent or multivalent antibodies on a single polypeptide chain, or to produce bispecific antibodies.

In other embodiments, additional modified antibodies can be made using nucleic acid molecules encoding the P-cadherin antibody. For example, "Kappa antibodies (Kappa diodes)" (Ill, et al, Protein Eng.10: 949-57(1997)), "miniantibodies (Minibodies)" (Martin, et al, EMBO J., 13: 5303-9(1994)), "diabodies" (Holliger, et al, Proc. Natl. Acad. Sci. USA, 90: 6444-.

Bispecific antibodies or antigen-binding fragments can be produced by a number of methods, including fusion of hybridomas or ligation of Fab' fragments. See, e.g., Songsivilai & Lachmann, clin. exp. immunol, 79: 315-: 1547-1553(1992). In addition, bispecific antibodies can be formed as "diabodies" or "Janusins". In some embodiments, the bispecific antibody binds to two different epitopes of P-cadherin. In some embodiments, a modified antibody as described above is made using one or more variable domains or CDR regions from a human P-cadherin antibody provided herein.

Vectors and host cells

To express the antibodies and antigen-binding portions of the invention, DNA encoding partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked into a vector such that transcriptional and translational regulatory sequences within the vector provide their intended function of regulating 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. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YAC, EBV-derived episomes, and the like. The antibody genes are linked into the vector such that transcriptional and translational regulatory sequences within the vector provide their intended function of regulating transcription and translation of the antibody genes. 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 may be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., linking the antibody gene fragment to complementary restriction sites on the vector, or to blunt ends in the absence of restriction sites).

A convenient vector is one which encodes a fully functional human C_HOr C_LImmunoglobulin sequences with appropriate restriction sites designed to allow for any V_HOr V_LSequences can be easily inserted and expressed, as described above. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also occurs in the presence of human C_HIn the splicing region in the exon. Polyadenylation and transcription termination are present in natural chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame 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 from a non-immunoglobulin protein).

In addition to the antibody chain gene, the recombinant expression vector of the present invention carries a regulatory sequence that regulates the expression of the antibody chain gene in a host cell. One skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on the following factors: for selection of the host cell to be transformed, the desired level of protein expression, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers from retroviral LTRs, promoters and/or enhancers from Cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), promoters and/or enhancers from simian virus 40(SV40) (e.g., SV40 promoter/enhancer), promoters and/or enhancers from adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma promoters, and strong mammalian promoters such as native immunoglobulin promoters and actin promoters. Further description of viral regulatory elements and their sequences is found, for example, in U.S. Pat. No.5,168,062, U.S. Pat. No.4,510,245, and U.S. Pat. No.4,968,615. Methods for expressing antibodies in plants (including descriptions of promoters and vectors) and methods for plant transformation are known in the art. See, for example, 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.

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

Nucleic acid molecules encoding the P-cadherin antibodies and vectors comprising these nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial or yeast host cells. Transformation can be performed by any known method for introducing a polynucleotide into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene (1, 5-dimethyl-1, 5-diaza-undecamethylene polymethine bromide) -mediated transfection, protoplast fusion, electroporation, liposome-encapsulated polynucleotides, and direct microinjection of DNA into the nucleus. Alternatively, the nucleic acid molecule may be introduced into a mammalian cell by a viral vector. Methods for transforming cells are well known in the art. See, for example, U.S. patent nos.4,399,216, 4,912,040, 4,740,461 and 4,959,455, incorporated herein by reference. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium (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 a number of 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, Baby Hamster Kidney (BHK) cells, African green monkey kidney Cells (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 cell lines with high expression levels. Other cell lines that may be used are insect cell lines such as Sf9 or Sf21 cells. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell, or more preferably, to allow the antibody to be secreted into the medium in which the host cell is cultured. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, tobacco, Arabidopsis, duckweed, maize, wheat, potato, and the like. Bacterial host cells include e. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

In addition, expression of the antibodies of the invention by the producer cell line can be enhanced using a number of known techniques. For example, glutamine synthetase (GS system) and DHFR gene expression systems are popular approaches for enhancing expression under certain conditions. High expressing cell clones can be identified using conventional techniques such as limiting dilution cloning and microdroplet techniques. The GS system is described in european patent nos.0216846, 0256055, 0323997 and 0338841.

It is likely that antibodies expressed by different cell lines or transgenic animals will have glycosylation that differ from one another. However, all antibodies encoded by the nucleic acid molecules provided herein, or all antibodies comprising the amino acid sequences provided herein, are encompassed by the present invention, whether or not the antibody is glycosylated.

Transgenic animals and plants

The P-cadherin antibodies of the invention can also be produced transgenically by producing mammals or plants which are genetically transformed with the immunoglobulin heavy and light chain sequences of interest and which produce the antibody in recoverable form. For transgenic production in mammals, P-cadherin antibodies can be produced in and recovered from the milk of a goat, cow, or other mammal. See, for example, U.S. patent nos.5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference.

In some embodiments, a non-human transgenic animal comprising a human immunoglobulin locus is immunized with P-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.

In some embodiments, a non-human transgenic animal or plant is produced by introducing one or more nucleic acid molecules of the invention (which encode a P-cadherin antibody or antigen-binding portion thereof) into the animal or plant by standard transgenic techniques. See Hogan and United States patent 6,417,429, supra. The transgenic cell used to make the transgenic animal may be an embryonic stem cell or a somatic cell or a fertilized egg. The transgenic non-human organism may be chimeric, heterozygote of non-chimeric, and homozygote of non-chimeric. See, for example, Hogan et al,Manipulating the Mouse Embryo：A Laboratory Manual，2^nd ed.，Cold Spring Harbor Press(1999)；Jackson，et al.，Mouse Genetics and Transgenics：A Practical Approachoxford university Press (2000); and a (d) a (n) kert,Transgenic Animal Technology：A Laboratory Handbookacademic Press (1999), which is incorporated herein by reference. In some embodiments, the transgenic non-human animal has targeted disruption and replacement of a targeting construct encoding a heavy and/or light chain of interest. P-cadherin can be produced in any transgenic animal. In preferred embodiments, the non-human animal is a mouse, rat, sheep, pig, goat, cow, or horse. Non-human transgenic animals express the encoded polypeptide in blood, milk, urine, saliva, tears, mucus, and other bodily fluids.

Class conversion

The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass (IgG1, IgG2, IgG3, or IgG4) of P-cadherin can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific for a particular antibody class and subclass. Such antibodies are commercially available. The class and subclass can be determined by ELISA or Western blotting, among other techniques. Alternatively, the class and subclass can be determined as follows: the species and subclasses of antibodies are determined by sequencing all or part of the constant regions of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to known amino acid sequences of a variety of immunoglobulin classes and subclasses. The P-cadherin antibodies of the invention may be IgG, IgM, IgE, IgA or IgD molecules. For example, the P-cadherin antibody may be an IgG that is of the IgG1, IgG2, IgG3, or IgG4 subclasses. In one embodiment, the P-cadherin antibody can have the amino acid sequence of SEQ ID NO: 344 and SEQ ID NO: 345, or a light chain constant region.

One aspect of the invention provides methods for converting a class or subclass of P-cadherin antibodies to another class or subclass. In some embodiments, the code V is isolated using methods well known in the art_LOr V_HThe nucleic acid molecule of (1), which does not comprise a coding C_LOr C_HThe sequence of (a). The nucleic acid molecule is then contacted with a nucleic acid molecule encoding C from the immunoglobulin class or subclass of interest_LOr C_HThe nucleic acid sequences of (a) are operably linked. This may be carried out using a carrier or comprising C_LOr C_HThe nucleic acid molecules of the strand are completed as described above. For example, P-cadherin antibodies, initially IgM, can be species-converted to IgG. In addition, class switching can be used to switch one IgG subclass to another, for example from IgG1 to IgG 2. Another method of producing an antibody of the invention comprising an isotype of interest comprises the steps of: isolating nucleic acids encoding the heavy chain of a P-cadherin antibody and nucleic acids encoding the light chain of a P-cadherin antibody, isolating nucleic acids encoding V_HSequence of region V_HThe sequences are linked to sequences encoding the constant domains of the heavy chains of the isoforms of interest, the light chain genes and heavy chain constructs are expressed in cells, and the P-cadherin antibodies with the isoforms of interest are collected.

Deimmunized antibodies

In another aspect of the invention, antibodies or antigen-binding portions thereof may be deimmunized to reduce their immunogenicity using techniques such as those described in PCT publication Nos. WO98/52976 and WO00/34317 (incorporated herein by reference).

Derivatized and labeled antibodies

The P-cadherins of the invention or antigen-binding portions thereof can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or portion thereof is derivatized such that P-cadherin binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody portions of the invention are intended to include the intact and modified forms of human P-cadherin described herein. For example, an antibody or antibody moiety of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent binding, or otherwise) to one or more other molecular moieties, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a tag, a cytotoxic agent, an agent, and/or a protein or peptide, which are capable of mediating binding of the antibody or antibody moiety to another molecule (e.g., a streptavidin nuclear region or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different type, e.g., to produce a bispecific antibody). Suitable cross-linking agents include hetero-bifunctional agents (e.g., m-maleimidobenzoxy-N-hydroxysuccinimide ester) having two different reactive groups separated by a suitable spacer region, or homo-bifunctional agents (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.

Another type of derivatized antibody is a labeled antibody. Detection agents that may be used to derive the antibodies or antigen-binding portions of the invention include fluorescent compounds (including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc.). The antibody may also be labeled with an enzyme suitable for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. Where the antibody is labelled with a detectable enzyme, it may be detected by the addition of an additional reagent with which the enzyme produces a reaction product which can be distinguished. For example, when horseradish peroxidase is present, the addition of catalase and diaminobiphenyl results in a colored reaction product, which is detectable. Antibodies can also be labeled with biotin and detected by indirect measurement of avidin or streptavidin binding. Antibodies can also be labeled with a predetermined polypeptide epitope that is recognized by a secondary reporter gene (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding domain, epitope tag). In some embodiments, the tags are joined by spacer arms of various lengths to reduce potential steric hindrance. P-cadherins can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups are useful for facilitating the biological properties of the antibody, such as increasing the half-life of the serum.

Affinity of P-cadherin antibodies for P-cadherin

Avidity (K) of P-cadherin antibodies or antigen-binding portions thereof for P-cadherin_D) And dissociation Rate (k)_off) Can be measured by methods known in the art. Can be measured by ELISA, RIA, flow cytometry or surface plasmon resonance, e.g. BIACORE^TMTo measure affinity. The dissociation rate can be measured by surface plasmon resonance. Preferably, the affinity and dissociation rate are measured by surface plasmon resonance. More preferably, BIACORE is used^TMThe affinity and dissociation rate were measured. Methods known in the art can be used to determine whether an antibody has substantially the same K as a P-cadherin antibody_D. Such an assay K_DAnd k_offThe method of (3) can be used in an initial screening phase, and in a subsequent optimization phase.

Identification of P-cadherin epitopes recognized by P-cadherin

The present invention provides human P-cadherin antibodies that bind to P-cadherin and compete or cross-compete with and/or bind to the same epitope as any of the antibodies described in table 1 or 2. Methods known in the art can be used to determine whether an antibody binds to the same epitope as a P-cadherin antibody of the invention, or cross-competes for binding. In one embodimentThe P-cadherin antibodies of the invention are allowed to bind to P-cadherin under saturating conditions, and the ability of the test antibody to bind to P-cadherin is then measured. The test antibody and the P-cadherin antibody bind different epitopes if the test antibody is capable of binding to P-cadherin simultaneously with the P-cadherin antibody. However, if the test antibody is not able to bind to P-cadherin at the same time, the test antibody binds to the same epitope or an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the human P-cadherin antibody. The experiment can use ELISA, RIA, BIACORE^TMOr flow cytometry. In a preferred embodiment, the experiment is performed using ELISA.

Inhibition of P-cadherin activity by P-cadherin antibodies

A number of assays can be used to identify P-cadherin antibodies that inhibit P-cadherin activity. For example, cell aggregation assays provide a means to measure P-cadherin-dependent cell aggregation. This type of assay uses a cell line that overexpresses P-cadherin, where the cells are placed in suspension and allowed to form P-cadherin-dependent aggregates. The ability of the P-cadherin antibody to prevent this aggregation is then quantified using an aggregation test by measuring the size of cell aggregates caused with and without the use of the antibody. The size of the cell aggregates as a function of the concentration of the P-cadherin antibody can then be used to determine IC₅₀The value is obtained. Example 3 provides methods for measuring several P-cadherin antibody ICs₅₀Additional details of the P-cadherin-dependent aggregate assay of values.

Cell adhesion assays can also be used to measure the ability of P-cadherin to block the adhesion of cells to receptor P-cadherin, which has been immobilized on a solid support. This type of assay can be performed, for example, by immobilizing P-cadherin on a solid support such as plastic. The cells overexpressing P-cadherin are then allowed to attach to a solid support via P-cadherin-P-cadherin interaction. The level of attachment can then be quantified with or without the use of the P-cadherin antibody. As a function of antibody concentration, the attachment can then be used to determine IC₅₀The value is obtained. Example 3 provides methods for measuring P-cadherin antibody IC₅₀Additional details of the P-cadherin-dependent cell attachment assay of values.

Inhibition of P-cadherin activity can also be measured using a P-cadherin-dependent spheroid destruction assay. This type of assay measures the ability of P-cadherin antibodies to disrupt preformed P-cadherin-dependent cell aggregation. By measuring the size reduction of aggregates (as a function of antibody concentration), the IC can be determined₅₀The value is obtained. Example 5 provides methods for measuring P-cadherin antibody IC₅₀Additional details of the P-cadherin-dependent spheroid disruption assay of values. The methods and assays described above for determining inhibition of P-cadherin activity by various antibodies or antigen-binding portions thereof can be used in an initial screening phase and subsequent optimization phase.

Molecular selectivity

The selectivity of the P-cadherin antibodies of the invention for other cadherins (e.g., E-cadherin) can be determined using methods known in the art. For example, the selectivity can be determined using Western blotting, flow cytometry, ELISA, immunoprecipitation, or RIA. Example 7 provides additional details of an ELISA assay for measuring the selectivity of a particular antibody for P-cadherin over E-cadherin. The methods and assays described above for determining the selectivity of various antibodies, or antigen-binding portions thereof, for P-cadherin can be used in an initial screening phase and subsequent optimization phase.

Pharmaceutical compositions and administration

The present invention also relates to a pharmaceutical composition for treating abnormal cell growth in a mammal, including a human, comprising an amount of a P-cadherin antibody, or antigen-binding portion thereof, as described herein, effective to treat abnormal cell growth, and a pharmaceutically acceptable carrier.

The antibodies and antigen binding portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antigen-binding portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically suitable. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered 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. Other examples of pharmaceutically acceptable substances are wetting agents or minor auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which prolong the shelf life or increase the effectiveness of the antibody.

The compositions of the present invention may be in a variety of forms, such as 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 mode of administration and therapeutic application to be employed. Typical preferred compositions are injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. Preferred modes of administration are 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 in, for example, ampoules or multi-dose containers, with or without an added preservative. The compositions may be in the form of, for example, 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.

Typically, therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the P-cadherin antibody in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which 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 of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Proper fluidity of the 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 dispersants) and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

The antibodies or antibody portions of the 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. One skilled in the art will appreciate that the route and/or mode of administration will vary depending on the desired result.

In certain embodiments, the antibody compositions of the invention can be prepared with carriers that will protect the antibody from rapid release, such as controlled release components, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are generally known to those skilled in the art. See, for exampleSustained and Controlled Release Drug DeliverySystems，J.R.RobinsonEd., marcel dekker, inc., New York, 1978, which is incorporated herein by reference.

Additional active compounds may also be incorporated into the compositions. In certain embodiments, the heterogeneous P-cadherin antibodies of the invention are co-formulated and/or co-administered with one or more additional therapeutic agents. These agents include, but are not limited to, antibodies that bind to other targets, anti-tumor agents, anti-angiogenic agents, signal transduction inhibitors, anti-proliferative agents, chemotherapeutic agents, or peptide analogs that inhibit P-cadherin. Such combination therapy may require low doses of inhibitory P-cadherin and co-administered agents, thereby avoiding possible toxicity or complications that accompany multiple monotherapies.

The compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding portion of the invention. By "therapeutically effective amount" is meant an amount effective, at a dosage or for a requisite period of time, to achieve the desired therapeutic result. The therapeutically effective amount of the antibody or antigen-binding portion can vary depending on factors such as the stage of the disease, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to respond as desired in the individual. A therapeutically effective amount is also an amount wherein a therapeutically beneficial effect outweighs any toxic or detrimental effect of the antibody or antigen binding portion. A "prophylactically effective amount" is an amount effective, at dosages or for periods of time necessary, to achieve the desired prophylactic result. Typically, because prophylactic doses are used in subjects prior to or at an early stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

The dosing regimen may be adjusted to provide the best desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus (bolus) may be administered, several divided doses may be administered over time or the dose may be scaled down or up as required by the treatment situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form to simplify administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for the mammalian subjects being treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the unit dosage form of the invention is dictated by and directly dependent on (a) the unique characteristics of the P-cadherin antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved; and (b) the inherent limitations of such antibodies in the field of formulation for use in the treatment of sensitivity in an individual.

An exemplary, non-limiting range of therapeutically or prophylactically effective amounts of an antibody or antibody portion of the invention is from 0.025 to 50mg/kg, more preferably from 0.1-25, 0.1 to 10, or 0.1 to 3 mg/kg. In some embodiments, the formulation contains 5mg/mL of the antibody in a buffer of 20mM sodium citrate (pH5.5), 140mM NaCl, and 0.2mg/mL polysorbate 80. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is also to be understood that for any particular subject, specific dosing regimens should be adjusted over time according to the individual need and the professional judgment of the person administering the compositions or administering the compositions, and that the dosage ranges disclosed herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Another aspect of the invention provides a kit comprising an antibody or antigen-binding portion of P-cadherin of the invention, or a composition comprising such an antibody or portion. In addition to the antibody or composition, the kit can include a diagnostic or therapeutic agent. The kit may include instructions for use in a diagnostic method or a therapeutic method. In a preferred embodiment, the kit comprises an antibody or a composition comprising an antibody, and a diagnostic agent useful in the methods described below. In another preferred embodiment, the kit comprises an antibody or a composition comprising an antibody, and one or more therapeutic agents useful in the methods described below.

Diagnostic methods of use

P-cadherin or antibody-binding portions thereof can be used in diagnostic methods to detect P-cadherin in biological samples in vitro or in vivo. For example, P-cadherin can be used in conventional immunoassays, including but not limited to ELISA, RIA, flow cytometry, tissue immunohistochemistry, Western blotting, or immunoprecipitation. The P-cadherins of the invention can be used to detect P-cadherins from humans. P-cadherins can also be used to detect P-cadherins from mice, rats and cynomolgus monkeys.

The present invention provides a method for detecting P-cadherin in a biological sample comprising contacting the biological sample with P-cadherin of the invention and detecting bound antibodies. In one embodiment, the P-cadherin antibody is directly labeled with a detectable label. In another embodiment, the P-cadherin antibody (primary antibody) is unlabeled and a secondary antibody or other molecule capable of binding the P-cadherin antibody is labeled. As is well known to those skilled in the art, a second antibody is selected that is capable of specifically binding to a particular species and class of first antibody. For example, if the P-cadherin antibody is human IgG, the second antibody may be anti-human-IgG. Other molecules capable of binding to antibodies include, but are not limited to, protein a and protein G, all of which are commercially available from, for example, Pierce Chemical co.

Suitable labels for the antibody or secondary antibody have been previously discussed and include a variety of enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive materials include¹²⁵I、¹³¹I、³⁵S or³H。

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

The above immunoassays can be used for a large number of purposes. For example, antibodies to P-cadherin can be used to detect P-cadherin in cultured cells. In a preferred embodiment, the P-cadherin antibody is used to detect the amount of P-cadherin produced by cells that have been treated with a plurality of compounds. The method can be used to identify compounds that modulate the level of P-cadherin. According to this method, one cell is treated with one test compound for a period of time while the other sample is not treated. If total P-cadherin levels are to be measured, the cells are lysed and the total P-cadherin levels are measured using one of the immunoassays described above. The total P-cadherin levels of treated cells versus untreated cells were compared to determine the effect of the test compound.

Preferred immunoassays for measuring total P-cadherin levels are flow cytometry or immunohistochemistry. Such as ELISA, RIA, flow cytometry, Western blotting, immunohistochemistry, cell surface labeling of membrane-resident proteins and immunoprecipitation are well known in the art. See, e.g., Harlow and Lane, supra. In addition, immunoassays can be improved to high throughput screening, thereby detecting the activity of a large number of compounds or inhibition of P-cadherin.

The P-cadherin antibodies of the invention may also be used to detect P-cadherin levels in a tissue or in cells derived from the tissue. In some embodiments, the tissue is diseased tissue. In some embodiments of the method, the tissue or tissue biopsy thereof is excised from the patient. The tissue or tissue biopsy is then used in an immunoassay to determine, for example, total P-cadherin levels or localized P-cadherin by the methods described above.

The antibodies of the invention are also useful in vivo for identifying tissues and organs that express P-cadherin. One advantage of using the human P-cadherin antibody of the invention is that: unlike antibodies of non-human origin or humanized or chimeric antibodies, they can be safely used in vivo without eliciting a significant immune response to the antibody as a result of administration.

The method comprises the following steps: administering a detectably labeled P-cadherin antibody or a composition comprising the same to a patient in need of such a diagnostic test, and performing image analysis on the patient to determine the location of the tissue expressing P-cadherin. Image analysis is well known in the medical arts and includes, but is not limited to, x-ray analysis, Magnetic Resonance Imaging (MRI), or Computed Tomography (CT). The antibody may be labelled with any agent suitable for in vivo imaging, for example a contrast agent such as barium (which may be used for x-ray analysis), or a magnetic contrast agent such as gadolinium chelates (which may be used for MRI or CT). Other labeling agents include, but are not limited to, radioisotopes, such as⁹⁹Tc. In another embodiment, the P-cadherin antibody may be unlabeled and may be imaged by administering a second antibody or other molecule that is detectable and capable of binding the P-cadherin antibody. In one embodiment, a tissue biopsy is obtained from the patient to determine whether the tissue of interest expresses P-cadherin.

Therapeutic methods of use

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

In another embodiment, the P-cadherin antibody, or antibody portion thereof, can be administered to a patient expressing abnormally high levels of P-cadherin. The antibody may be administered once, but more preferably is administered multiple times. The antibody may be administered three times a day to every six months or more. Administration can be according to the following schedule: three times a day, two times a day, once every two days, once every three days, once a week, once every two weeks, once a month, once two months, once three months, and once six months. The antibody may also be administered continuously by a micropump. The antibody may be administered by mucosal, buccal, intranasal, inhalational, intravenous, subcutaneous, intramuscular, parenteral, or intratumoral routes. The antibody may be administered once, at least twice, or at least for a period of time until the condition is treated, alleviated, or cured. Generally, the antibody is administered as long as the condition exists. Typically, the antibody will be administered as part of a pharmaceutical composition as described above. Generally, the antibody dose will be in the range of 0.1 to 100mg/kg, more preferably 0.5 to 50mg/kg, more preferably 1 to 20mg/kg and even more preferably 1 to 10 mg/kg. Serum antibody concentrations can be measured by any method known in the art.

The present invention also relates to a method for treating abnormal cell growth in a mammal (including a human) comprising administering to said mammal a therapeutically effective amount of P-cadherin or an antigen binding portion thereof, which is effective in treating abnormal cell growth, as described above.

In one embodiment of the method, the abnormal cell growth is a cancer, including, but not limited to, mesothelioma, hepatobiliary (liver and bile duct) cancer, primary or secondary CNS tumor, primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, epidermal or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and duodenal) cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, lymphoblastic lymphoma, chronic myelogenous leukemia, and other cancers of the liver and biliary tract, Bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, Central Nervous System (CNS) neoplasms, primary CNS lymphoma, non-hodgkins's lymphoma, spinal axis tumors (spinal axis tumors), brain stem glioma, pituitary adenoma, adrenal cortex cancer, single-capsule cancer, multiple myeloma, bile duct cancer, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

In a preferred embodiment of the invention, the cancer is selected from lung cancer (NSCLC and SCLC), head and neck cancer, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, breast cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, Central Nervous System (CNS) neoplasms, primary CNS lymphoma, non-hodgkins's lymphoma, spinal axis tumors, or a combination of one or more of the foregoing cancers.

In a preferred embodiment of the invention, the cancer is selected from lung cancer (NSCLC and SCLC), ovarian cancer, colon cancer, rectal cancer, cancer of the anal region or a combination of one or more of the above.

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

The present invention also relates to a method for treating abnormal cell growth in a mammal comprising administering to the mammal an amount of a P-cadherin antibody, or an antigen-binding portion thereof, effective to treat abnormal cell growth, as described herein, and an anti-neoplastic agent selected from the group consisting of: inhibitors of nuclear division, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxins, anti-hormones, and anti-androgens.

The present invention also relates to a pharmaceutical composition for treating abnormal cell growth in a mammal (including a human) comprising an amount (as described herein, said amount being effective to treat abnormal cell growth) of P-cadherin, or an antigen-binding portion thereof, in combination with a pharmaceutically acceptable carrier and an anti-neoplastic agent selected from the group consisting of: inhibitors of nuclear division, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, and anti-androgens.

The present invention also relates to methods for treating a hyperproliferative disease in a mammal comprising administering to said mammal a therapeutically effective amount of a P-cadherin antibody, or an antigen-binding portion thereof (as described herein), and an anti-neoplastic agent selected from the group consisting of: antiproliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-I inhibitors, cox-II inhibitors, nuclear division inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxins, anti-hormones, statins, and anti-androgens.

In one embodiment of the invention, the antineoplastic agent used in combination with the P-cadherin antibody, or antigen-binding portion thereof, and the pharmaceutical composition described herein is an anti-angiogenic agent, a kinase inhibitor, a pan kinase inhibitor, or a growth factor inhibitor. Preferred pan kinase inhibitors include SU-11248 described in U.S. Pat. No.6,573,293(Pfizer, Inc, NY, USA).

Anti-angiogenic agents, including but not limited to, agents such as EGF inhibitors, EGFR inhibitors, VEGF inhibitors, VEGFR inhibitors, TIE2 inhibitors, IGFIR inhibitors, COX-II (cyclooxygenase II) inhibitors, MMP-2 (matrix-metalloproteinase 2) inhibitors, and MMP-9 (matrix-metalloproteinase 9) inhibitors. Preferred VEGF inhibitors include anti-VEGF monoclonal antibodies such as Avastin (bevacizumab) and Genentech, inc.

Other 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 octasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(Cytran Inc. of Kirkland, Washington, USA); and angiozyme- -synthetic synthases from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California), and combinations thereof. VEGF inhibitors useful in the practice of the present invention are disclosed in US patent nos.6,534,524 and 6,235,764, the entire contents of which are incorporated herein for all purposes. Particularly preferred VEGF inhibitors include CP-547,632, AGl3736, Vatalanib, Macugen, and combinations thereof.

Other VEGF inhibitors are described, for example, in WO99/24440 (published 20 May 1999), PCT International application PCT/IB99/00797 (filed 3 May 1999), WO95/21613 (published 17 August 1995), WO99/61422 (published 2 Decy 1999), U.S. Pat. No.6,534,524 (published 13736), U.S. Pat. No.5,834,504 (published 10 May 1998), WO98/50356 (published 12 May 1998), U.S. Pat. No.5,883,113 (published 16 Sanyue 1999), U.S. Pat. No.5,886,020 (published 23 Sanyue 1999), U.S. Pat. No. US6,653,308 (published 11 August 2003), WO99/10349 (published 4 May 1999), WO 97/321997 (published 12), WO97/22596 (published 1997), WO 544626/5484 (published 1998)/856 Decyule 1998 (published 1998), WO98/02438 (published 22 January 1998), WO99/16755 (published 8 January 1999), and WO98/02437 (published 22 January 1998), the entire contents of which are incorporated herein by reference.

Other antiproliferative agents which may be used with the antibodies or antigen-binding portions thereof of the present invention include enzyme inhibitors of farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFr, including the compounds disclosed and claimed in the following U.S. patent applications: 09/221946 (filed on December 28 of 1998); 09/454058 (filed on December 2 of 1999); 09/501163 (February 9, 2000 submission); 09/539930 (March 31, 2000); 09/202796 (submitted on May 22 days 1997); 09/384339 (filed on august 26 days 1999) and 09/383755 (filed on august 26 days 1999); and the compounds disclosed and claimed in the following U.S. provisional patent applications: 60/168207 (filed 30 november 1999); 60/170119 (filed 10 December 1999); 60/177718 (submitted in january 21 of 2000); 60/168217 (filed 30/november 1999) and 60/200834 (filed 1/may 2000). The entire contents of each of the above-mentioned patent applications and provisional patent applications are incorporated herein.

Pdgfr inhibitors include, but are not limited to, those disclosed in: WO01/40217, published on July 7 in 2001, and WO2004/020431, published on March 11 in 2004, the contents of which are incorporated herein in their entirety for all purposes. Preferred PDGFr inhibitors include Pfizer's CP-673,451 and CP-868,596 and pharmaceutically acceptable salts thereof.

Preferred GARF inhibitors include Pfizer's AG-2037 (pellitrexol and pharmaceutically acceptable salts thereof). GARF inhibitors suitable for use in the practice of the present invention are disclosed in U.S. patent No.5,608,082, which is incorporated in its entirety for all purposes.

Examples of useful COX-II inhibitors that may be used in combination with the P-cadherin antibodies or antigen-binding portions thereof described herein and the pharmaceutical compositions described herein include: CELEBREX^TM(celecoxib), parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189(Lumiracoxib), BMS347070, RS57067, NS-398, Bextra (valdecoxib), paracib, Vioxx (rofecoxib), SD-8381, 4-methyl-2- (3, 4-dimethylphenyl) -1- (4-sulfamoyl-phenyl) -1H-pyrrole, 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoyl-phenyl) -1H-pyrrole, T-614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125, and Arcoxib (etoricoxia).Additionally, COX-II inhibitors are disclosed in U.S. patent application nos.10/801,446 and 10/801,429, the contents of which are incorporated in their entirety for all purposes.

In a preferred embodiment, the antineoplastic agent is celecoxib as disclosed in U.S. patent No.5,466,823, the contents of which are incorporated by reference in their entirety for all purposes. The structure of celecoxib is shown below:

in a preferred embodiment, the antineoplastic agent is faricib (valecoxib) as disclosed in U.S. patent No.5,633,272, the contents of which are incorporated by reference in their entirety for all purposes. The structure of the valicoxib is shown below:

in a preferred embodiment, the antineoplastic agent is parecoxib as disclosed in U.S. patent No.5,932,598, the contents of which are incorporated by reference in their entirety for all purposes. The structure of parecoxib is shown below:

in one embodiment, the antineoplastic agent is deracoxib as described in U.S. patent No.5,521,207, the contents of which are incorporated by reference in their entirety for all purposes. The structure of deracoxib is shown below:

in a preferred embodiment, the antineoplastic agent is SD-8381 as disclosed in U.S. patent No.6,034,256, the contents of which are incorporated by reference in their entirety for all purposes. The structure of SD-8381 is shown below:

in a preferred embodiment, the antineoplastic agent is ABT-963 as disclosed in international publication No. WO2002/24719, the contents of which are incorporated by reference in their entirety for all purposes. The structure of ABT-963 is shown below:

in a preferred embodiment, the antineoplastic agent is rofecoxib as shown below:

in a preferred embodiment, the antineoplastic agent is MK-663 (etoricoxib), as disclosed in International publication No. WO1998/03484, the contents of which are incorporated by reference in their entirety for all purposes. The structure of etoricoxib is shown below:

in a preferred embodiment, the antineoplastic agent is COX-189(Lumiracoxib) as described in International publication No. WO1999/11605, the contents of which are incorporated by reference in their entirety for all purposes. The structure of Lumiracoxib is shown below:

in a preferred embodiment, the antineoplastic agent is BMS-347070 as disclosed in us patent No.6,180,651, the contents of which are incorporated by reference in their entirety for all purposes. The structure of BMS-347070 is as follows:

in a preferred embodiment, the antineoplastic agent is NS-398(CAS 123653-11-2). The structure of NS-398(CAS123653-11-2) is shown below:

in a preferred embodiment, the antineoplastic agent is RS57067(CAS 17932-91-3). The structure of RS57067(CAS17932-91-3) is shown below:

in a preferred embodiment, the antineoplastic agent is 4-methyl-2- (3, 4-dimethylphenyl) -1- (4-sulfamoyl-phenyl) -1H-pyrrole. The structure of 4-methyl-2- (3, 4-dimethylphenyl) -1- (4-sulfamoyl-phenyl) -1H-pyrrole is shown below:

in a preferred embodiment, the antineoplastic agent is 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole. The structure of 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole is shown below:

in a preferred embodiment, the antineoplastic agent is meloxicam (meloxicam). The structure of meloxicam is shown below:

other useful inhibitors for use as anti-tumor agents with the antibodies of the invention and the pharmaceutical compositions described herein include aspirin and non-carrier anti-inflammatory drugs (NSAIDs), which inhibit the enzymes that make prostaglandins (cyclooxygenase I and II), resulting in lower prostaglandin levels, including but not limited to the following: salsalate (Amigesic), fluorophenylsalicylic acid (diffisil) (Dolobid), Ibuprofen (ibuprophen) (Motrin), Ketoprofen (Ketoprofen) (oridis), Nabumetone (Nabumetone) (Relafen), Piroxicam (Piroxicam) (Feldene), Naproxen (Naproxen) (Aleve, Naprosyn), Diclofenac (Diclofenac) (voltar), Indomethacin (Indomethacin) (Indocin), Sulindac (Sulindac) (Clinoril), Tolmetin (Tolmetin) (Tolectin), Etodolac (Etodolac) (Lodine), Ketorolac (Ketorolac) (torolol), Oxaprozin (oxypzin) (Daypro), and combinations thereof. Preferred COX-I inhibitors include ibuprofen (Motrin), sulfamethoxazole, naproxen (Aleve), indomethacin (Indocin), nabumetone (Relafen), and combinations thereof.

Targeting agents for use in combination with the P-cadherin antibodies or antigen-binding portions thereof described herein and the pharmaceutical compositions described herein include EGFr inhibitors such as Iressa (Iressa) (gefitinib), AstraZeneca (AstraZeneca)), Tarceva (Tarceva) (erlotinib) or OSI-774, OSI Pharmaceuticals (Inc.), and bicistribux (cetuximab, impact Pharmaceuticals, Inc.), EMD-7200(Merck AG), ABX-EGF (Amgen Inc. and Abgenix Inc.), HR3(Cuban gorment), IgA antibodies (universities of langen-number), TP-38 (ivegf), EGFr fusion proteins, liposome-vaccines, anti-EGFr immune (hernices) and combinations thereof.

Preferred EGFr inhibitors include iressa, dibepredx, dereprednol and combinations thereof. The invention also relates to an anti-tumor agent selected from the group consisting of pan erb receptor inhibitors and ErbB2 receptor inhibitors, such AS CP-724,714(Pfizer, Inc.), CI-1033 (Canatinib), Pfizer, Inc.), Heng ai Ping (Herceptin) (trastuzumab, Genentech Inc.), Omita (Omitarg) (2C4, pertactin (pertuzumab), Genentech), TAK-165(Takeda), GW-572016 (lonafarnib), GlaxoSmithine, GW-282974 (GlaxoSmithine), EKB-569(Wyeth), PKI-166(Novartis), dHER2(HER2 Vaccine, Corixa and GlaxoSmithKline), HER 38724 (HER2 Vaccine, Dendren 2/neurone (HER 2/neurone), and Klaxon-3 (Biogene), and bispecific antibody (Biogene), and specific antibody combinations thereof, such AS mAb-39209, and Biogene, and specific antibody (antibiotic, such AS Biogene, Inc, and Biogene, Inc, and specific antibody, such AS Streptococcus, Klaxon-282974, Klaxon, Klebsiel, Kluy, and Biogene, and antibody. Preferred erb selective antineoplastic agents include Herceptin, TAK-165, CP-724,714, ABX-EGF, HER3 and combinations thereof. Preferred pan erbb receptor inhibitors include GW572016, CI-1033, EKB-569, and omitave and combinations thereof.

Other erbB2 inhibitors include those described below: WO98/02434 (published 22 January 1998), WO99/35146 (published 15 January 1999), WO99/35132 (published 15 January 1999), WO98/02437 (published 22 January 1998), WO97/13760 (published 17 January 1997), WO95/19970 (published 27 January 1995), U.S. Pat. No.5,587,458 (published 24 January 1996) and U.S. Pat. No.5,877,305 (published 2 January 1999), each of which is incorporated herein by reference in its entirety. Inhibitors of the ErbB2 receptor suitable for use in the present invention are also described in U.S. patent nos.6,465,449 and 6,284,764 and international application No. wo2001/98277, each of which is incorporated herein by reference in its entirety.

In addition, the additional antineoplastic agent may be selected from the following: BAY-43-9006(OnyxPharmaceuticals Inc.), Gentiangseis (Genasense) (Augmerosen, Genta), Pannithermam (Panitumumab) (Abgenix/Amgen), Jewel (Zevalin) (Schering), Toxico mo (Bexxar) (Corixa/GlaxoSmithKline), Abarelix (Abarelix), force ratio Tai (Alimta), EPO 906(Novartis), Descemenolide (discotide) (XAA-296), ABT-510(Abbott), cancerol (Neocastat) (Aema), Enzesterlin (Enzastaurin) (Elarizly), windmill seed antibiotic (Combrratin) A4 (4P (Oxigen-XA), Ashmerin (Astinene) (Availand/Availant), Availanthus (Availanthus) (Availa/Availand combination thereof), Availanthus (Availa/Avicula), Availand (Avicula) and Avicula (Avicula, and Avicula, Ab, and Avicula, Ab, Abies, Ab.

Other antineoplastic agents may be selected from the following: cyprotene (cyproterone acetate), histrelin (Histerelin acetate), Piranix (Plenaixis) (abarelipt), Atrasentan (Atrasentan) (ABT-627), Satraplatin (Satraplatin) (JM-216), thalidomide (thalidomide), Serratide (Theratope), timilene (Temilifene) (DPPE), ABI-007 (paclitaxel), Evepitant (Eva) (raloxifene), Atamestane (Atamestane) (Biomed-777), Polychitax (Xtax, glutamamatate paclitaxel), Tagetin (targetine) (bezatriptane), and combinations thereof.

Additionally, the other antineoplastic agent may be selected from the following: texadone (trizalone) (tirapazamine), epsipramine (Aposyn, exisulind), napthalwitt (Nevastat) (AE-941), chromatography forest (Ceplene) (histamine dihydrochloride), rubitecan (orathenin, rubitecan), veegum (Virulizin), pancreatic cancer vaccine (gastimone) (G17DT), DX-8951f (irinotecan mesylate), Onconase (ranase), BEC2 (mitramumab), motetralin gadolinium (xtaxafin, motaxafin gadolinium), and combinations thereof.

Other antineoplastic agents may be selected from the following: biovar (CEA), neoteresin (NeuTrexin), and combinations thereof. The additional antineoplastic agent may be selected from the following agents: oraurin (OvaRex) (oregovomab), oxidey (Osidem) (IDM-1) and combinations thereof. The additional antineoplastic agent may be selected from the following agents: advexin (ING201), tirazyne (Tirazone) (tirapazamine), and combinations thereof. The additional antineoplastic agent may be selected from the following agents: RSR13 (ethiprole (efaproxiral)), Krtara (Cotara) (131I chTNT 1/b), NBI-3001(IL-4), and combinations thereof. The additional antineoplastic agent may be selected from the following agents: canvaxin, GMK vaccine, PEG Interon a, taxotere (Taxoprexin) (DHA/paclitaxel), and combinations thereof. Other preferred antineoplastic agents include the MEK1/2 inhibitor of Pfizer PD325901, the MEK inhibitor of Array Biopharm ARRY-142886, the CDK2 inhibitor of Bristol Myers BMS-387,032, the CDK inhibitor of Pfizer PD0332991, and the AXD-5438 of AstraZeneca, and combinations thereof. Additionally, mTOR inhibitors may also be utilized, for example, CCI-779(Wyeth) and rapamycin derivatives RAD001(Novartis) and AP-23573(Ariad), HDAC inhibitor SAHA (Merck inc./aton pharmaceuticals), and combinations thereof. Additional antineoplastic agents include the aurora 2 inhibitor VX-680(Vertex), Chk1/2 inhibitor XL844 (Exilixis).

One or more of the cytotoxic agents, for example selected from the group consisting of epirubicin (elence), docetaxel (Taxotere), paclitaxel, dexrazoxane (Zinecard), rituximab (Rituxan), imatinib mesylate (imatinib mesylate) (Gleevec), and combinations thereof, can be used in combination with the P-cadherin antibodies or antigen-binding portions thereof described herein and the pharmaceutical compositions described herein.

The invention also concerns the use of the antibodies and antigen-binding portions thereof of the invention with hormone therapies including, but not limited to, exemestane (amonasin, Pfizer Inc.), leuprolide (Lupron or leuprolin, TAP/Abbott/Takeda), anastrozole (Arimidex, Astrazeneca), gosrelin (Zoladex, Astrazeneca), doxercol, fadrozole, formestane, tamoxifen citrate (tamoxifen, Nolvadex, Astrazeneca), concanadex (Astrazeneca), Abarelix (Abarelix) (Praecis), protriline (Trelstar), and combinations thereof.

The present invention also relates to hormonal therapy agents such as antiestrogens including, but not limited to, fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole (Femara, Novartis); antiandrogens, e.g. bicalutamide, flutamide, mifepristone, nilutamide, Casodex(4 '-cyano-3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3' - (trifluoromethyl) propionylaniline, bicalutamide) and combinations thereof.

In addition, the invention provides an antibody of the invention, alone or in combination with one or more supportive care products, such as a product selected from sargrastim (Filgestim) (Neupogen), ondansetron (Zofran), Framacchia (Fragmin), erythropoietin (Procrit), Allerxi (Aloxi), emetine (Emend), or a combination thereof.

Particularly preferred cytotoxic agents include irinotecan (Camptosar), dibenz, Iressa, Gleevec, Taxotere (Gleevec), Taxotere (Taxotere), and combinations thereof.

The following topoisomerase I inhibitors can be used as anti-tumour agents: camptothecin, irinotecan hydrochloride (irinotecan HCl, irinotecan (Camptosar)), entecacin (edotecarin), rubitecan (orathecin) (Supergen), exatecan (exatecan, Daiichi), BN-80915(Roche), and combinations thereof. Specifically preferred topoisomerases II include epirubicin (elence).

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

Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan, dibromomannitol, carboquinone, thiotepa, ramustine, nimustine, temozolomide, AMD-473, hexamethylmelamine, AP-5280, apaziquone (apaziquone), bronosine (brosplastallicin), bendamustine, carmustine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, phosphoramide, and dibromodulcitol; platinum-complexed alkylating compounds include, but are not limited to, cisplatin, barbiturate (Paraplatin) (carboplatin), eletriplatin, lobaplatin, nedaplatin, oxaliplatin (Eloxatin, oxaliplatin, Sanofi), or satraplatin (satraplatin), and combinations thereof. Particularly preferred alkylating agents include oxaliplatin (Eloxatin).

Antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine, 5-fluorouracil (5-FU) alone or in combination with folinic acid (leucovorin), tegafur, UFT, doxifluridine, carmofur, cytarabine (cytarabine), cytarabine (cytarabine ocfosfate), enocitabine, S-1, pertaine (alimata, premerexed disodium, LY231514, MTA), Gemzar (Gemcitabine, Eli Lilly), fludarabine, 5-azacitidine, capecitabine, cladribine, clofarabine (clofarabine), decitabine, eforinine, ethynylcythemine (ethinylcytidine), cytarabine (cytarabine), hydroxyurea, TS-1, melphalan, larabine, norflurazone, trexasteine (octotrexate), pentetrexene (doxorhexetidine), litoridine (doxepidine), litorixate (doxepidine), doxepidine (doxepidine), ritin (doxepidine), litorine (doxepidine), ritin (doxepidine), trex (trex), trex (trexate), trex (trex, A combination of trimetrexate, vidarabine, vincristine, vinorelbine; or for example one of the preferred antimetabolites is disclosed in European patent application No.239362, such as N- (5- [ N- (3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl) -N-methylamino ] -2-thenoyl) -L-glutamic acid and combinations thereof.

Antibiotics include intercalating antibiotics but are not limited to: doxorubicin (aclarubicin), actinomycin d (actinomycin d), amrubicin (amrubicin), liposomal anthracycline (annamycin), doxorubicin (adriamycin), bleomycin (bleomycin), daunorubicin (daunorubicin), doxorubicin (doxorubicin), elsamitrucin (elsamitrumicin), epirubicin (epirubicin), galarucin (galarubicin), idarubicin (idarubicin), mitomycin C, nemorubicin (nemorubicin), neocarzinostatin (neocarzinostatin), pelomycin (pelyprolicin), pirarubicin (pirarubicin), butterfly mycin (rebeccamycin), staylamil (stimalacin), streptozocin (strezocin), amylrubicin (valrubicin), sethoxyrubicin (sethoxyrubicin), and stanin (stanin), and combinations thereof.

The antitumor substance derived from a plant includes, for example, a substance selected from the group consisting of a nuclear division inhibitor, such as vinblastine (vinblastine), docetaxel (Taxotere), paclitaxel (paclitaxel), and a combination thereof.

The cytotoxic topoisomerase inhibitor comprises one or more agents selected from the group consisting of: alreuptacin (aclarubicin), amonafide (amonafide), leupepton (belotecan), camptothecin (camptothecin), 10-hydroxycamptothecin (10-hydroxycamptothecin), 9-aminocampothecin (9-aminocampothecin), daptomycin (difitocan), irinotecan hydrochloride (Camptosar), edotecan, epirubicin (Ellence), etoposide (etoposide), exatecan (exatecan), piperikang (giecacan), lurtotecan (lurtothecan), mitoxantrone (mitoxantrone), pirarubicin (pirarubicin), pioglitazone (pixantrone), rubitecan (robuzone), SN-38, tylon (topotecan), topotecan (topotecan), and combinations thereof.

Preferred cytotoxic topoisomerase inhibitors include one or more agents selected from the group consisting of: camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan hydrochloride (Camptosar), entecacin (epotecarin), epirubicin (Ellence), epipodophyllotoxin glucopyranoside, SN-38, topotecan, and combinations thereof.

Immunizing agents include interferon and a number of other immune enhancers. Interferons include interferon alpha, interferon alpha-2 a, interferon alpha-2 b, interferon beta, interferon gamma-1 a, interferon gamma-1 b (activine), or interferon gamma-n 1, and combinations thereof. Other agents include filgrastim (filgrastim), lentinan (lentinan), schizophyllan (sizofilan), TheraCys, ubenimex (ubenimex), WF-10, aldesleukin (aldesleukin), alemtuzumab (alemtuzumab), BAM-002, dacarbazine (dacarbazine), daclizumab (daclizumab), dinil interleukin (denileukin), gemumab (gemtuzumab) ozogamicin (ozogamicin), imomab (ibritumomab), imiquimod (imiquimod), legumine (lentiograstim), lentinan (lentinan), melanoma vaccine (melaoma vacine) (Corixa), molestan (molgrastimastim), covax-CL, sargrastimastim (sargrastimastim), interleukin (melanogastine), interleukin (e.g., (rituximab), rituximab (e), lentinus (e), lentigine (e), e-e), e (e-e.g., E-E (e-E (E-E (E-, Prasuviger (Provenge) (Dendreon) and combinations thereof.

Biological response modifiers are agents that modify in vivo defense mechanisms or biological responses (e.g., survival, growth, or tissue cell differentiation) such that they have anti-tumor activity. Such agents include coriolus versicolor polysaccharide (krestin), lentinan (lentinan), sizopyran (sizopyran), streptolysin (picibanil), ubenimex (ubenimex) and combinations thereof.

Other anti-cancer agents include alitretinoin (alitretinin), polymyoside (ampigien), atrasentan (atrasentan) besartan (bezantene), bortezomib (bortezomib), busentan (Bosentan), calcitriol (calcerol), etisulin (exisulindle), finasteride (finasteride), fotemustine (fotemustine), ibandronic acid (ibandronic acid), miltefosine (miltefosine), mitoxantrone (mitoxantrone), 1-asparaginase, procarbazine (procarbazine), dacarbazine (dacarbazine), hydroxyurea (hydroxyurea), peiperidazine (pegavargase), pentostatin (pentitan), procarbazine (zatine), miltitant (teltaine) (tlvet, teltaine), and combinations thereof.

Other anti-angiogenic compounds include acitretin (acitretin), fenretinide (fenretinide), thalidomide (thalidomide), zoledronic acid (zoledronic acid), angiostatin (angiostatin), dehydrotunicin B (aplidine), cloxistine (cilengt), combretastatin A-4(combretastatin A-4), endostatin (endostatin), halofuginone (halofuginone), rebimastat (rebimastat), ramofluoride (removab), lenalidomide (revlimmid), squalamine (squalamine), ukraine (ukrain), monoclonal antibody LM609(Vitaxin), and combinations thereof.

Compounds complexed with platinum include, but are not limited to, cisplatin, carboplatin, nedaplatin, oxaliplatin, and combinations thereof.

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

Other antineoplastic agents include mitoxantrone, 1-asparaginase, procarbazine, dacarbazine, hydroxyurea, pentostatin, tretinoin, and combinations thereof.

Anti-tumor agents capable of enhancing anti-tumor immune responses such as CTLA4 (cytotoxic lymphocyte antigen 4) and other agents capable of blocking CTLA4 may also be utilized, for example, the CTLA4 compounds disclosed in MDX-010(Medarex) and U.S. Pat. No.6,682,736; and antiproliferative agents such as other inhibitors of farnesyl protein transferase, e.g. inhibitors of farnesyl protein transferase. In addition, specific CTLA4 antibodies useful in the present invention include those described in U.S. provisional application 60/113,647 (filed on december 23 of 1998), U.S. patent No.6,682,736, both of which are incorporated herein by reference in their entirety.

Specific IGF1R antibodies useful in the present invention include those described in international patent application No. wo2002/053596, which is incorporated herein by reference in its entirety.

Specific CD40 antibodies useful in the present invention include those described in international patent application No. wo2003/040170, which is incorporated herein by reference in its entirety.

Gene therapy agents may also be used as antineoplastic agents, such as tnfrede (genevec), which express TNF α in response to radiotherapy.

In one embodiment of the invention, statins may be used in combination with the P-cadherin antibodies or antibody binding portions thereof and pharmaceutical compositions thereof described herein. Statins (HMG-CoA reductase inhibitors) may be selected from Atorvastatin (Atorvastatin, Lipitor, Pfizer Inc.), pravastatin (Provastatin, Bristol-Myers Squibb), Lovastatin (Lovastatin, Mevacor, Merck Inc.), Simvastatin (simvasstatin, Zocor, Merck Inc.), Fluvastatin (Fluvastatin, Lescol, Novartis), schrivastatin (Cerivastatin, Baycol, Bayer), Rosuvastatin (Rosuvastatin, cretstor, aszeneca), Lovastatin (Lovastatin), and nicotinic acid (Niacin, Advicor, kos), derivatives thereof, or combinations thereof.

In a preferred embodiment, the statin is selected from atorvastatin and lovastatin, derivatives thereof or combinations thereof.

Other agents suitable for use as antineoplastic agents include amlodipine besylate/atorvastatin calcium (Caduet).

For any of the methods of treating a hyperproliferative disease or abnormal cell growth described herein, which use a P-cadherin antibody or antigen binding portion in combination with at least one additional therapeutic agent, the P-cadherin antibody can be conjugated to, or derivatized with, the additional therapeutic agent. The at least one additional therapeutic agent may also be administered alone, 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 may be administered in the same pharmaceutical formulation as the antibody, or it may be administered in a separate formulation.

Gene therapy

Nucleic acid molecules encoding the antibodies and antibody portions of the invention can be administered to a patient in need thereof by gene therapy. The therapy may be in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding both the heavy and light chains are administered to a patient. In a more preferred embodiment, the nucleic acid molecules are administered such that they are stably integrated into the chromosome of the B cell, since these cells are specialized for the production of antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and 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 for gene therapy include liposomes, plasmids and viral vectors. Exemplary viral vectors are retroviruses, adenoviruses and adeno-associated viruses. Following infection in vivo or ex vivo, antibody expression levels are monitored by sampling from the treated patient and using any immunoassay known in the art or described herein.

In a preferred embodiment, the gene therapy method comprises the steps of: administering an isolated nucleic acid molecule encoding a P-cadherin antibody heavy chain or an antigen-binding portion thereof, and expressing the nucleic acid molecule. In another embodiment, the gene therapy method comprises the steps of: administering an isolated nucleic acid molecule encoding a P-cadherin antibody light chain or an antigen-binding portion thereof, and expressing the nucleic acid molecule. In a more preferred embodiment, the gene therapy method comprises the steps of: administering an isolated nucleic acid molecule encoding a P-cadherin antibody heavy chain or an antigen-binding portion thereof and an isolated nucleic acid molecule encoding a P-cadherin antibody light chain or an antigen-binding portion thereof, and expressing the nucleic acid molecules. The gene therapy method may further comprise the step of administering another therapeutic agent (e.g., any of the agents previously described in connection with combination therapy).

In order that the invention may be better understood, the following examples are disclosed. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Examples

In the examples and formulations that follow, "BSA" refers to bovine serum albumin; "EDTA" means ethylenediaminetetraacetic acid; "DMSO" refers to dimethyl sulfoxide; "MOPS" refers to 3- (N-morpholino) propanesulfonic acid; "MES" refers to 2- (N-morpholino) methanesulfonic acid; "PBS" refers to phosphate buffered saline; "dPBS" refers to Dulbecco's phosphate buffered saline; "HEMA" refers to 2-hydroxy-ethyl methacrylate; "DMEM" refers to Dulbecco's modified eagle's Medium (eagle's medium); "FBS" means fetal bovine serum; "NEAA" refers to a nonessential amino acid; "HEPES" means N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid; "DMF" refers to dimethylformamide.

Example 1: screening scFv phage display libraries

Recombinant human P-cadherin (R & D Systems 861-PC-100) was used as an antigen to screen scFv phage display libraries. The large scFv human antibody library cloned into a phagemid vector was used for selection (Vaughan, T.J.et al, nat. Biotech.14: 309-314 (1996)). The scFv recognizing P-cadherin were isolated from phage display libraries in a series of repeated selection cycles taken for fusion monolayers of recombinant human P-cadherin and HCT116 cells expressing P-cadherin. Briefly, after incubation with the library, bound phage are recovered from the P-cadherin and unbound phage are washed away. Then as in Vaughan, t.j.et al, nat.biotech.14: 309, 314(1996) the bound phage were recovered and the selection process repeated. A representative proportion of clones from the selection cycle output were subjected to phage enzyme-linked immunosorbent assay (ELISA) to detect binding to P-cadherin, essentially as described by Vaughan, t.j.et al, nat.biotech.14: 309, 314 (1996). Two different antigens were used in the ELISA: recombinant human P-cadherins (R & D Systems) and A431 cells expressing P-cadherins. ELISA-positive clones were subjected to methods such as Vaughan, t.j.et al, nat.biotech.14: 309-: 293-302 (1998). Unique ELISA-positive clones were converted to intact IgG molecules and tested for their ability to neutralize P-cadherin in the P-cadherin-dependent adhesion assay described in example 4. Based on the results of this screen, antibody 129-1c4 (IC 50 of 1-3 μ M in a431 adhesion assay) was selected as the lead parental lineage for further optimization.

Example 2: pilot optimization

Mutagenesis of antibody variable heavy chains (V) by oligonucleotide guidance_H) And variable light chain (V)_L) The CD3 region was used to construct a phage display library from 129-1c 4. Using a process such as Clackson and Lowman,Phage Display-A Practical Approachthe library was constructed using standard molecular biology techniques as described in Oxford University Press 2004. Thereby performing affinity-based selection; after incubation with the library, recombinant human P-cadherin (R)&D Systems) were captured by G-protein coated paramagnetic beads (Dynal 100.03), bound phage were recovered by magnetic separation, while unbound complexes were washed away. The selection process was repeated, with decreasing concentrations of recombinant human P-cadherin present at the time of selection (25 nM to 10pM in 4 rounds). In addition, from V_HCDR3 and V_LThe selection output of the CDR3 library was recombined into another phage display library and selected in two additional rounds of affinity-based selection. A representative proportion of clones from the selection cycle output were screened as scFv in the 129-1c4 epitope competition assay described in example 8.

Example 3: p-cadherin-dependent adhesion assay

Determination of IC Using several optimized scFv in a P-cadherin-dependent adhesion assay Using the following protocol₅₀Values, the optimized scFv was converted to IgG in the antibody recovery optimization stage as described above in example 2. Average measurement IC of these antibodies₅₀The values are shown in table 4 below.

24 hours before use, 2mM CaCl was added₂The MilliQ aqueous solution of (2) recombinant human P-cadherin Fc (R)&D Cat.861-PC) was reconstituted to a concentration of 1mg/mL and stored at 4 ℃. A431 cells were cultured and prepared as follows. Conventionally, A431 cells (ECACC No.85090402) were cultured in Minimal Essential Medium (MEM) (Invitrogen Cat 31095) in Nunc triple flasks (3X175 cm)²Area), the minimal essential medium contains 10% fetal bovine serum (Invitrogen cat.10100-147) and 1% non-essential amino acids (Invitrogen cat.11140-035). Cultured cells were approximately 80% confluent when harvested for assay. To prevent possible passage-related eagles, cells harvested between passage 4 and passage 8 after 48 or 72 hours of culture are routinely used. A431 cells were harvested with 0.25% trypsin/1 mM EDTA (Gibco Cat 25200-056) for just enough time for cell detachment (7-10 min) and immediately diluted to about 2X10 in tissue culture medium⁶Density of cells/mL. A431 cells were then centrifuged (1200rpm), resuspended in assay buffer (Hanks Balanced salt solution (Mg-free)²⁺And Ca²⁺Phenolsulfonphthalein-free) (Invitrogen Cat.14175-053) supplemented with 1mM final CaCl₂Final concentration), recentrifuge and re-at 2x10⁶The final concentration of cells/mL was resuspended in assay buffer.

Preparation of IgG serial dilutions and preincubation with a431 cells was performed as follows. 180 μ L of each test IgG or portion thereof was supplemented with 20 μ L of assay buffer containing 10% BSA (Sigma Cat. A-9576) and the BSA concentration was normalized to 1%. Anti-murine/human P-cadherin was initially referred to as a polyclonal antibody (R)&D cat. af-761) were resuspended in MilliQ water to give a 1mg/mL stock solution. Then 40. mu.L of this stock was further diluted into 140. mu.L of assay buffer. Then 20. mu.L of assay buffer containing 10% BSA was added to give 200. mu.L of solution (0.2mg/mL antibody and 0.1% BSA). Duplicate serial dilutions were prepared by first adding 2x90 μ L of AF-761 polyclonal antibody or detection IgG to the first column of Greiner 96-well polypropylene dilution plates (Greiner cat.780271). Then 60. mu.L of assay buffer was added to columns 2-11. Dilutions of 30. mu.L to 60. mu.L (1: 3) were then prepared on the plate from columns 1-11, left to right. Then to the hole12A-D60. mu.L of assay buffer was added separately to define the maximum adhesion. Minimal adhesion was defined by adding 60 μ L of 25mM EDTA (in assay buffer) to wells 12E-H. Then 60. mu.L of A431 cell suspension (2X 10 in assay buffer) was added to all wells⁶cells/mL-prepared as underlined above), plates were preincubated at 37 ℃ for 1 hour after agitation.

Preparation of test IgG serial dilutions and preincubation with a431 cells, P-cadherin coated assay plates were prepared as follows. Recombinant human P-cadherin Fc in coating buffer (Mg-free)²⁺And Ca²⁺PBS-Invitrogen Cat.14190-094) to a concentration of 10. mu.g/mL and dispensed onto Fluoron unc 96(Nunc Cat.437958) well assay plates (100. mu.L/well). The plates were then incubated at room temperature for 1 to 30 minutes. The plates were then washed 3 times with PBS using a Tecan 96 plate washer. Then 200. mu.L/well of assay buffer was added for blocking and the plate was incubated at room temperature for an additional 1 hour. The plates were then washed 3 times with PBS as before.

100 μ L/well of pre-incubated IgG/A431 material was then transferred from the Greiner 96-well dilution plate to a P-cadherin coated assay plate. The IgG/a431 material was mixed by pipetting during transfer to ensure that the cells were homogeneous. The adhesion process was then allowed to occur by incubating the assay plate for 30-45 minutes at 37 ℃. At the end of incubation, non-adherent cells were removed by gentle aspiration of the medium from the plate and cell wash buffer (supplemented to 1mM CaCl)₂Final concentration of Hanks Balanced salt solution (Mg free)²⁺And Ca²⁺And no phenolsulfonphthalein-Invitrogen cat.14175-053) refill wells. The plate was then placed upside down in a bath of cell wash buffer for 15 minutes to remove residual, non-adherent cells. At the end of this incubation, the contents of the wells were gently aspirated.

The quantification of adherent cells was performed as follows. Adherent cells were detected by adding 100. mu.L/well of a combined lysis/alkaline phosphatase detection reagent (diethanolamine substrate buffer 5 Xconcentrate diluted 1: 5 with water (Pierce Cat.34064), followed by dissolving one 15mg tablet of PNPP (SigmaCat. N-2640) per 25mL of 1 Xsolution and then incubating for 30-60 minutes at 37 ℃. The reaction was then stopped by adding 1M NaOH (50. mu.L/well) to give a maximum OD value of about 0.8 in the absence of inhibition. The absorbance at 405nm was then measured using a standard plate reader.

The results were then analyzed as follows. Column 12, wells a-D as 100% adhesion and columns 12, wells E-H as 0% adhesion, the raw data is first converted to% adhesion values as follows:

% adhesion { (adhesion value-minimum adhesion value)/(maximum adhesion-minimum adhesion) } 100

IC was then determined using Prism software to calculate% adherence ratio IgG inhibitor concentration₅₀The value is obtained. When partial inhibition is observed, IC₅₀Quoted as the concentration of IgG that produces a true 50% inhibition compared to the midpoint of the curve. IC (integrated circuit)₅₀The values are reported in table 4.

TABLE 4

IgG	A431 adhesion determination of average IC50(nM)(n＝3)
		194-e06	0.162
194-a02	0.217
		194-b09	0.229
195-e11	0.114
		194-g09	0.158
196-h02	0.148
		194-e01	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

Two independent a431 adhesion assays were performed and several germlined, optimized iggs from the 129-1c4 lineage were studied compared to their non-germlined equivalents. For these experiments, a new batch of P-cadherin (CFR-1340) had to be changed41) IC showing slight improvement over other data₅₀The values are correlated. The average data from these two experiments for several germlined iggs is shown in table 5. As previously described, g-194-b09 refers to a germlined version of 194-b09, and so on.

TABLE 5

IgG	A431 adhesion determination of 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-e11	0.87
195-e11	0.97
		g-200-h06	1.31
200-h06	0.63

Example 4: p-cadherin-dependent cell aggregation assay

The following protocol was used for determining IC in a P-cadherin-dependent aggregation assay₅₀Value, the method uses several optimized scfvs that are converted to IgG in the antibody recovery optimization stage as described in example 2 above. Because cell lines that overexpress P-cadherin form tight multicellular aggregates when placed in suspension growth, cell aggregation can be measured in the presence of P-cadherin antibodies that affect cell aggregation. Average measurement IC of several antibodies₅₀Shown in table 6 below.

Plates were prepared as follows. Each 96-well assay plate was coated with 50 μ L of polyhema (12mg/mL in 90% ethanol, 10% methanol) and then evaporated for 6 hours overnight before washing with 3 × 100 μ L sterile H2O before use. The cells were then cultured as follows. From the human cell line SW480 (stably expressing P-cadherin G418)^r) The cells of (2) were passaged in complete growth medium (qs DMEM (Invitrogen 11995-. The cultures were then frozen in growth medium + 10% DMSO.

Day 1 at 5x10⁶Cells/100 mm plate, dilution no greater than 1: 3 inoculated SW 480: pCAD cells and control SW 480: pCLNX (stable control vector). The cells were then grown in culture for 48 hours. About 10x10 per 100mm plate⁶Or enough cells for a 2x96 well plate.

On day 3, the medium was removed and washed with dPBS (Dulbecco's PBS (InVitrogen 14040) -133), after which the cells were trypsinized in 3mL/100mm dishes. Neutralization was performed after release of complete growth medium in two volumes (6 mL). The plate was then washed three times using a 10mL pipette to disrupt the clusters. Cells were then counted and pellets obtained using a Beckman centrifuge at 1000rpm for 5 minutes. The medium was aspirated, and the pellet was first resuspended in < 1mL of complete growth medium by finger vortexing followed by p1000 pipette pipetting, and then the cell concentration was normalized to 1.3M/mL. The dispersion of individual cells was ensured by microscopy.

Reagent plates were then prepared by blocking 96-well plates with dPBS and 5% PBS for 30 minutes. The plates were then washed with 1 × 100 μ L of pbs followed by aspiration and flicking to dryness. Using 4x [ IgG ]]Concentration, a dilution series of test IgG was prepared with dPBS, which was sufficient to treat 3 wells of the plate in one of the 96 wells. 4000 in 30. mu.L/well0 cells were aliquoted into 96-well, washed, poly-HEMA-coated Costar3590 non-tissue culture plates (Corning 3590). Then 10 μ L of reagent was transferred to each well of the 96-well plate. Each treatment of triplicate samples was performed using an 8 channel pipette. Then shaken at 250rpm at 37 ℃ in the presence of moisture and 5% CO₂Incubate overnight (16-18 hours).

On day 4, 40. mu.L of the shaken cells were transferred to a polylysine-coated 96-well plate (BioCoat polylysine-coated 96-well plate: BD 356516). The wells were then washed with 60 μ L of complete growth medium, the plates were shaken by tapping, and transferred to plates coated with polylysine. An additional 50 μ L wash was performed as needed. Followed by a wet 37 ℃ C, 5% CO₂Incubate for 60 minutes in the incubator. All cells were carefully transferred in number at this step, as gently as possible, without undue pipetting. Cells were then fixed by adding 100 μ L of fixative (7.4% formaldehyde (37% wt/vol. -Sigma F15587)) in a fume hood followed by incubation at room temperature for > 30 minutes.

To wash the cells, the liquid was then decanted into a collection cup or dish and flicked to remove residual liquid, gently poured onto a paper towel. Then 100 μ L of pbs per well was added for washing, followed by incubation for 15 minutes. Cells were then stained by decanting as above and using 100 μ L of Hoescht (1 g/mLHoescht-Hoescht 10mg/mL Molecular Probes 33342 in dPBS), followed by incubation for 30 min. The cells were then washed twice, leaving 100 μ L of pbs in the wells for microscopy.

The number of objects aggregated per well (Cellomics) was then measured, and the mean object count (test IgG) was compared to IgG (e.g., Gt-anti-P-cadherin R)&D Systems AF761) or a control of medium alone. The concentration of the IgG inhibitor in the ratio of the object count to the object count is then calculated and the IC is determined₅₀The value is obtained. IC of several antibodies of the invention₅₀The values are reported in table 6.

TABLE 6

IgG	SW480 aggregation assay mean IC50(nM))
		194-e06	0.7
194-a02	1.1
		g-194-b09	0.9
g-195-e11	2.2
		g-194-g09	2.6
196-h02	3.2
		194-e01	1.3
196-d10	1.5
		g-196-g03	0.9
196-e06	1.9
		195-a09	1.7
198-a09	2.9
		g-200-h06	4.7
129-1c4	35

Example 5: p-cadherin-dependent spheroid destruction assay

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

Plates were prepared as follows. Each 96-well assay plate was coated with 50. mu.L of poly-HEMA (90% ethanol, 12mg/mL in 10% methanol), then evaporated for 6 hours overnight before use with 3X 100. mu.L sterile H₂And O washing. The cells were then cultured as follows. From the human cell line SW480 (stably expressing P-cadherin G418)^r) The cells of (2) were passaged in complete growth medium (qs DMEM (Invitrogen 11995-. Then freezing the culture in a growth cultureNutrient + 10% DMSO.

Day 1 at 5x10⁶Cells/100 mm plate, dilution no greater than 1: 3 inoculated SW 480: pCAD cells and control SW 480: pCLNX (stable control vector). The cells were then grown in culture for 48 hours. About 10x10 per 100mm plate⁶Or enough cells for a 2x96 well plate.

On day 3, the medium was removed and washed with dPBS (Dulbecco's PBS (InVitrogen 14040) -133), after which the cells were trypsinized in 3mL/100mm dishes. Neutralization was performed after release of complete growth medium in two volumes (6 mL). The plate was then washed three times using a 10mL pipette to disrupt the clusters. Cells were then counted and pellets obtained using a Beckman centrifuge at 1000rpm for 5 minutes. Aspirate the medium, resuspend the pellet first in < 1mL of complete growth medium by finger vortexing followed by p1000 pipette pipetting, then normalize the cell concentration to 1.0x10⁶and/mL. The dispersion of individual cells was ensured by microscopy.

40000 cells in 40. mu.L/well were aliquoted into 96-well, washed, poly-HEMA-coated Costar3590 non-tissue culture plates (Corning 3590). Then shaken at 250rpm at 37 ℃ in the presence of moisture and 5% CO₂Incubate overnight (16-18 hours).

On day 4, reagent plates were prepared by blocking 96-well plates with dPBS and 5% PBS for 30 minutes. The plates were then washed with 1 × 100 μ L of pbs followed by aspiration and flicking to dryness. Using 5x [ IgG ]]Concentration, a dilution series of test IgG was prepared with dPBS, which was sufficient to treat 3 wells of the plate in one of the 96 wells. Each treatment of triplicate samples was performed using an 8 channel pipette. Then shaken at 250rpm at 37 ℃ in the presence of moisture and 5% CO₂Culturing in an incubator (20-24 hours).

On day 5, 50. mu.L of the shaken cells were transferred to a polylysine-coated 96-well plate (BioCoat polylysine-coated 96-well plate: BD 356516). The wells were then washed with 50 μ L of complete growth medium, the plates were shaken by tapping, and transferred to plates coated with polylysine. Need to make sure thatAn additional 50. mu.L wash was performed. Followed by a wet 37 ℃ C, 5% CO₂Incubate for 60 minutes in the incubator. All cells were carefully transferred in number at this step, as gently as possible, without undue pipetting. Cells were then fixed by adding 100 μ L of fixative (7.4% formaldehyde (37% wt/vol. -Sigma F15587)) in a fume hood followed by incubation at room temperature for > 30 minutes.

To wash the cells, the liquid was then decanted into a collection cup or dish and flicked to remove residual liquid, gently poured onto a paper towel. Then 100 μ L of pbs per well was added for washing, followed by incubation for 15 minutes. Cells were then stained by decanting as above and using 100 μ L of Hoescht (1 g/mLHoescht-Hoescht 10mg/mL Molecular Probes 33342 in dPBS), followed by incubation for 30 min. The cells were then washed twice, leaving 100 μ L of pbs in the wells for microscopy. The number of objects aggregated per well (Cellomics) was then measured, and the mean object count (test IgG) was compared to IgG (e.g., Gt-anti-P-cadherin R)&D Systems AF761) or a control of medium alone. The concentration of the object count ratio IgG inhibitor was then calculated to determine IC₅₀The value is obtained. Alternatively, the object count is expressed as the fold of destruction compared to the control at one of the determined concentrations shown in table 7.

TABLE 7

IgG	SW480 spheroid disruption assay fold increase at 5nM compared to control
		194-e06	10
194-a02	10
		g-194-b09	10
g-195-e11	7
		g-194-g09	14
196-h02	10
		194-e01	16
196-d10	10
		g-196-g03	13
196-e06	10
		195-a09	10
198-a09	13
		g-200-h06	7
129-1c4	4

Example 6: measurement of K of P-cadherin antibody_DAnd k_off

Using the manufacturer's protocol, BIACORE was used as follows^TM3000 Equipment affinity measurement of P-cadherin scFv Single-chain antibodies by surface plasmon resonance (K)_DAnd k_off)。

For kinetic analysis, recombinant human P-cadherin/Fc fusion protein (hCad/Fc) and mouse P-cadherin/Fc fusion protein (mCad/Fc) were immobilized on CM5 BIACORE using conventional amine coupling^TMOn a separate flow cell of the sensor chip. Using a catalyst containing 2.0mM CaCl₂The 10mM acetate buffer of (9) pH4.5 was used as a fixation buffer to prepare the surface and to achieve protein densities of 5800 and 1600RU for hCad/Fc and mCad/Fc fusion proteins, respectively. The deactivation of the unreacted N-hydroxysuccinimide ester was carried out using 1M ethanolamine hydrochloride, pH 8.5. Activated/inactivated blank surfaces were used as control surfaces. Samples of scFv antibodies were prepared in running buffer at concentrations ranging from 200nM to 0.78nM (including 0nM solution containing running buffer only as zero reference). Samples were randomized and injected in triplicate through the flow cell for 1 min using HBS-P (10mM HEPES pH7.4, 150mM NaCl, 0.005% Surfactant P20) with 2.0mM CaCl₂As an electrophoresis buffer. The affinity constant was determined using a flow rate of 25. mu.L/min. Antibody dissociation was detected for 5 min and the surface was regenerated by 12 sec injection of 10mM glycine-HCl pH1.5 (25. mu.L/min). Raw data using Scrubber (BioLogic Software) Software package processing using CLAMP ((II)BioLogic Software) Software package analysis. Data as a wholeConforms to the Langmuir binding model of 1: 1.

Table 8 lists the affinity constants of the single chain anti-P-cadherin antibodies of the present invention:

TABLE 8

scFv	hCad/FcKD(nM)	hCad/Fckoff(1/s)	mCad/FcKD(nM)	mCad/Fckoff(1/s)
					194-b09	4.0	1.9x10-03	11	3.6x10-3
194-g09	2.6	1.6x10-03	1.8	8.1x10-4
					196-g03	1.1	7.0x10-04	0.76	4.7x10-4

Example 7: determination of the selectivity of P-cadherin antibodies

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

Recombinant human P-cadherin (R)&D Systems 861-PC-100) and recombinant human E-cadherin (R)&D Systems 648-EC-100) at 1. mu.g/mL in PBS +0.5mM CaCl₂Middle was coated on the wells of an Exiqon protein fixation plate (VWR International). Sample IgG at 3% Marvel/PBS +0.5mM CaCl before titration from 50nM (7.5. mu.g/mL) to 0.64nM (0.096. mu.g/mL)₂Medium blocking for 1 hour and adding in duplicate to wells coated with two different antigens. After overnight equilibration at 4 ℃, plates were plated with 1 xPBS/0.1% Tween +0.5mM CaCl₂Washed three times, then with 1xPBS +0.5mM CaCl₂Washed three times. Then 50. mu.L of 1: 5000 diluted in 3% Marvel/PBS +0.5mM CaCl was added to each well₂The anti-human Fab peroxidase conjugate in (a) was allowed to equilibrate for 1 hour at room temperature. Plates were plated with 1 xPBS/0.1% Tween +0.5mM CaCl₂Washed three times with 1xPBS +0.5mM CaCl₂Washed three times. To each well was added 50. mu.l of 3,3 ', 5, 5' -tetramethylbenzidine (TMB; Sigma), the reaction was allowed to proceed for 20 minutes, after which time the reaction was allowed to proceed by adding 25. mu.L/well 0.5M H₂SO₄The reaction was terminated. After reading the absorbance values at 450nm, the data were analyzed using Graphpad Prism software to calculate the relative K for each antibody binding to P-cadherin and E-cadherin_DThe value is obtained. The data obtained are summarized in table 9.

TABLE 9

IgG	P-cadherin KD(pM)	E-cadherin KD(pM)	KD(E)/KD/(P)
				194-a02	116	682	6
g-194-b09	788	2156	3
				g-194-g09	2602	Is not combined with	＞100
194-e01	112	20511	183
				g-194-e06	191	794	4
195-a09	120	11516	96
				194-e06	56	264	5
196-d10	45	63	1.4
				196-e06	62	187	3
g-196-g03	449	7513	17
				196-h02	102	33212	326
198-a09	78	19396	249

Example 8: epitope competition assay

IC was measured in epitope competition assays using the following protocol₅₀Values to measure the ability of variant scFv in the parental lineage to replace native P-cadherin on the surface of a431 cells with parent IgG. The amount of bound biotinylated parent IgG in the presence of inhibitory scFv was determined using europium streptavidin (europeium streptoavidin) and DELFIA quantification. Measured IC of several scFv₅₀The values are shown in table 10.

A431 cells (ECACC No.85090402) cultured according to standard methods and subsequently harvested at about 80% confluence were plated at 2.5X10 the day before use⁴Cells were seeded per well in 96-well Beckton Dickenson (BD Cat.6407) collagen-coated plates. This was followed by immersion in a PBS buffer (Gibco cat.14190-094, without calcium, magnesium and sodium bicarbonate) vessel, followed by addition of 200 μ L per well of blocking buffer (PBS Gibco cat.14190-094, plus 3% Marvel (premier international Foods Ltd.)) and incubation at room temperature for 2 hours. The plate was then washed 3 times with PBS as above.

For high throughput screening and IC₅₀For mapping, the scFv/IgG material was first prepared in a total volume of 60 μ L of assay buffer in a Greiner dilution plate (Greiner 96 well polypropylene plate (Greiner cat.780271)). Then 50 μ L was transferred directly from the controlled Greiner plate to the assay plate and the binding reaction was allowed to proceed for 2 hours 30 minutes at room temperature. At the end of the binding reaction, the plates were washed three times in PBS, after which europium-labeled streptavidin (Perkin Elmer Cat.1244-360, 1: 1000 diluted in DELFIA assay buffer (Perkin Elmer Cat.4002-0010) was added and incubated at room temperature for an additional 1 hour.

The plates were then washed 7 times with DELFIA wash buffer (Perkin Elmer Cat.4010-0010) by repeatedly immersing the plates in a buffer container. Finally, after the addition of DELFIA enhancer (Perkin Elmer Cat.4001-0010-.

Greiner dilution plate Assembly-HTS

High Throughput Screening (HTS) conditions are set up differently depending on the phase of optimization. For a subject V_HAnd V_LChain-optimized output HTS, finally [ peri-prep]Was set to 12.5% such that the parent scFv gave partial inhibition. For a source from V_H∶V_LLate HTS output of the recombinant library, the final peri-prep concentration was reduced to 1.7% under these conditions via V_HThe optimized baseline scFv (TOP-108-C01) gave partial inhibition. Optimization of peri-prep concentrations resulted in partial inhibition of the relevant baseline scFv, which established a window for identification of improved clones.

To achieve these final scFv peri-prep concentrations, the following steps were taken:

i) the desired volume (see below) of peri-prep sample material was transferred from the deep well sample plate to Greiner dilution plate columns 1-11 using the Cybiwell apparatus.

[ Final peri-prep ] transfer volume

[12.5％]＝7.5uL

[ 1.7% ] ═ 10.0uL (peri-prep diluted 1: 10)

(1: 10 Pre-diluted peri-prep was prepared by transferring 10. mu.L of pure peri-prep from the sample plate to a Greiner dilution plate containing 90. mu.L of assay buffer (using Cybiwell))

ii) the volume of columns 1-11 is then made up to 30 μ L by adding the appropriate volume of assay buffer.

iii) then 30. mu.L of assay buffer was added to column 12 (A-D) (fully bound wells).

iv) column 12 (E-H) was added 30. mu.L of excess unlabeled parent IgG (129-1c4) in assay buffer to define nonspecific binding. Was added at a concentration of 1000nM to give a final concentration of 500 nM.

v) biotinylation of 129-1c4 IgG using the water soluble reagent EZ-link-NHS-LC-Biotin (Perbio/Pierce product No. 21336). By adding 1/10 volumes of 1M NaHCO₃And 1/10 volumes of dimethylamide to supplement the IgG solution. EZ-link-NHS-LC-Biotin reagent was dissolved in DMF and then added in a molar excess of five times IgG, and the reaction was allowed to proceed for 20 min at room temperature. Biotinylated IgG was then stabilized by addition of BSA (0.1%). Then to all wells 30 μ L of biotinylated parent 129-1c4 IgG in assay buffer was added to give a final concentration of 1.5nM in a final 60 μ L volume (i.e. europium-labeled IgG was added at 2x final concentration).

vi) after mixing the contents of the Greiner plates by stirring, 50 μ Ι _ were directly transferred to the assay plates, allowing the binding reaction to proceed for 2 hours 30 minutes at room temperature (as described previously).

Greiner dilution plate Assembly-IC₅₀Drawing

i) 30 μ L/well assay buffer was added to columns 2-11 and wells 12A to 12D of a standard Greiner dilution plate.

ii) 30 μ L of excess unlabeled 129-1c4 parent IgG in assay buffer was added to column 12 (E-H) to define non-specific binding. This should be added at a concentration of 1000nM to give a final concentration of 500 nM.

iii) Add 2X 45. mu.L of each undiluted scFv His-prep to column 1, enabling 4 duplicate 11-point ICs per 96-well assay plate₅₀And (4) titrating. A1: 3 two-part serial dilution was then performed by taking 15. mu.L from column 1 and mixing with column 2, followed by taking 15. mu.L from column 2 and mixing into column 3, and so on. After mixing into column 11, 15. mu.L was removed (i.e., a final volume of 30. mu.L remained).

iv) 30 μ L of biotinylated 129-1c4 parent IgG in assay buffer was then added to all wells to give a final concentration of 1.5nM in a final 60 μ L volume (i.e. europium-labeled IgG was added at 2x final concentration).

v) after mixing the contents of the Greiner plate by stirring, 50. mu.L was transferred directly to the assay plate, allowing the binding reaction to proceed for 2 hours 30 minutes at room temperature as described previously.

For HTS, scFv was expressed in 96-well plates as a crude extract from bacterial periplasmic membrane (scFv peri-prep) in basal buffer containing MOPS/EDTA/Sorbitol pH7.4(MES pH 7.4). Just IC₅₀For mapping, scFv were expressed in a lower flux purified form using scFv His-tag (scFv His-prep) PBS in base buffer for affinity purification.

For HTS and IC₅₀For both analyses, the raw data was first converted to% bound data according to the following equation:

% binding { (binding value-non-specific binding)/(total binding-non-specific binding) } 100

HTS data was then further analyzed using standard Excel templates to identify cues that gave greater inhibition (lower% binding) compared to the relevant baseline scFv. To IC₅₀For analysis,% binding data was analyzed using Prism version 4.0 curve fitting software. ScFv is a variant of the 129-1c4 parent IgG, where V_HAnd V_LThe CDR3 regions were randomly mutated as described in example 2. V_HThe CDR3 variant sequence is shown in fig. 1 as SEQ ID NOs: 91 to 256. V_LThe CDR3 variant sequence is shown in fig. 1 as SEQ ID NOs: 257 to 319. IC of several scFv showing improved binding over the parent 129-1c4 IgG₅₀The values are shown in table 10. Each scFv variant of the 129-1c4 parent is represented by two sequences SEQ ID NOs: v_HCDR3 and V_LCDR3 identification. For reference, the IC of the parent 129-1c4 IgG₅₀Was 108.3 nM.

Watch 10

SEQ ID NO：VH CDR3	SEQ ID NO：VL CDR3	IC50(nM)
			37	40	2.49
37	261	1.78
			37	43	5.67
37	274	8.73
			37	277	2.20
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	304	1.10
37	305	2.19
			95	47	2.36
97	47	1.33
			113	47	2.06
125	47	0.26
			131	47	0.35
31	47	2.05
			147	47	1.16
165	47	5.11
			167	47	0.60
180	47	0.43
			181	47	2.46
27	47	0.54
			195	47	1.29
187	43	1.58
			27	296	1.16
163	43	2.45
			26	40	0.76
26	43	0.93
			27	309	1.36
199	310	2.54
			201	310	2.26
207	291	2.73

240

313

2.98

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that: certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

Claims (12)

1. An isolated antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion binds to P-cadherin and comprises:

a) as shown in SEQ ID NO: 24 disclosed V_H CDR1；

b) As shown in SEQ ID NO: 25 of the disclosure_H CDR2；

c) As shown in SEQ ID NO: 29 of the disclosure V_H CDR3；

d) As shown in SEQ ID NO: 38 of the publication V_L CDR1；

e) As shown in SEQ ID NO: 39 to thereby implement a chemical mechanical polishingV of_LA CDR 2; and

f) as shown in SEQ ID NO: 44 of the disclosure_L CDR3。

2. The antibody or antigen binding portion thereof of claim 1, wherein V is_HThe structural domain is shown as SEQID NO: shown at 321.

3. The antibody or antigen binding portion thereof of claim 1, wherein V is_LThe structural domain is shown as SEQID NO: 327.

4. The antibody or antigen binding portion thereof of claim 1, wherein V is_HThe domain comprises SEQ ID NO: 321, and V_LThe domain comprises SEQ ID NO: 327, or a sequence disclosed herein.

5. An isolated nucleic acid comprising SEQ ID NO: v disclosed in 321_HDomain and SEQ ID NO: 327 of_LAn antibody or antigen binding portion thereof of a domain.

6. The antibody of claim 5, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 344, wherein the light chain constant region comprises SEQ ID NO: 345, and SEQ ID NO: 344 is optionally cleaved.

7. An isolated P-cadherin antibody, or antigen-binding portion thereof, wherein the antibody is g-194-g09, V of the antibody g-194-g09_HAnd V_LThe amino acid and DNA sequence of SEQ id no: 321. SEQ ID NO: 327 and SEQ ID NO: 333. SEQ ID NO: 339 are described.

8. A pharmaceutical composition comprising the antibody or antigen-binding portion of claim 1 and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to claim 7 and a pharmaceutically acceptable carrier.

10. An isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof according to any one of claims 1-7.

11. An isolated nucleic acid molecule encoding a polypeptide as set forth in SEQ ID NO: 321, or a fragment thereof.

12. As shown in SEQ ID NO: 333.