HK1098160B - A ca6 antigen-specific cytotoxic conjugate and methods of using the same - Google Patents

Description

CA6 antigen-specific cytotoxic conjugates and methods of use thereof

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application 60/488,447, filed 21/7/2003.

Technical Field

The present invention relates to murine anti-CA 6 glycoepitope (glycope) monoclonal antibodies and humanized or resurfaced versions thereof. The invention also relates to epitope-binding fragments of the anti-CA 6 saccharide epitope monoclonal antibody, as well as to epitope-binding fragments of humanized or resurfaced versions of the anti-CA 6 saccharide epitope monoclonal antibody.

The invention further relates to cytotoxic conjugates comprising a cell-binding agent and a cytotoxic agent, therapeutic compositions comprising the conjugates, methods of using the conjugates to inhibit cell growth and treat disease, and kits comprising the cytotoxic conjugates. In particular, the cell binding agent is a monoclonal antibody or epitope-binding fragment thereof that recognizes and binds to a CA6 glycotope or a humanized or resurfaced version thereof.

Background

There have been many attempts to develop anti-cancer therapeutics that need to specifically target cancer cells without harming surrounding non-cancer cells and tissues. Such therapeutic agents have the potential to greatly improve the treatment of cancer in human patients.

One promising approach is to link cell binding agents such as monoclonal antibodies to cytotoxic drugs (Sela et al, in Immunoconjugates 189-216(C. Vogel, ed.1987); Ghose et al, in Targeted drugs 1-22(E. Goldberg, ed.1983); diene et al, in Antibody mediated delivery systems1-23(J. Rodwell, ed.1988); Pietryz et al, in Antibody mediated delivery systems 25-53(J. Rodwell, ed.1988); Bumol et al, in Antibody mediated delivery systems 55-79(J. Rodwell, ed.1988). depending on the choice of cell binding agent, these cytotoxic agents can be designed to bind only to specific cancer cell types based on the expression of the molecule expressed on the surface of such cells.

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been used in cytotoxic conjugates that are linked to various murine monoclonal antibodies. In some cases, the drug molecule is linked to the antibody molecule by an intermediate carrier molecule, such as serum albumin (Garnett et al, 46 Cancer Res.2407-2412 (1986); Ohkawa et al, 23 Cancer Immunol.h-nmother.81-86 (1986); Endo et al, 47Cancer Res.1076-1080(1980)), dextran (Hurwitz et al, 2 Appl.biochem.25-35 (1980); Manabi et al, 34 biochem.Pharmacol.289-291 (1985); Dillman et al, 46 Cancer Res.4886-4891 (1986); Shoval et al, 85Proc.Natl.Acad.Sci.8276-8280(1988)), or polyglutamic acid (Cheuka et al, 73. Natl.721.721.721.721-729, 1984. 19814, 19814 J.19814-1602 (1985); Tsuad et al, 1984. 19814).

Examples of specific conjugates that have shown some promise are the conjugate of the C242 antibody to CanAg, an antigen expressed on colorectal and pancreatic tumors, and maytansine-derived DM1 (Liu et al, Proc Natl Acad Sci USA, 93: 8618-8623 (1996)). Evaluation of the conjugate in vitro showed a high binding affinity for CanAg expressed on the cell surface, with an apparent K_dValue of 3X 10^-11M, and its cytotoxic effect on CanAg positive cellsHigh force (tension), IC thereof₅₀Is 6 x 10^-11And M. This cytotoxicity is antigen-dependent because the cytotoxic effect is blocked by an excess of unconjugated antibody and because antigen-negative cells are more than 100-fold less sensitive to the conjugate. Other examples of antibody-DM 1 conjugates with high affinity for the respective target cells and high antigen-selective cytotoxicity include conjugates of huN901, which is a humanized form of anti-human CD56 antibody; a conjugate of huMy9-6 that is a humanized form of an anti-CD 33 antibody; a conjugate of huC242 that is a humanized form of an antibody against the CanAg Muc1 epitope; huJ591, which is a deimmunized antibody (deimmunized antibody) against PSMA; trastuzumab, which is a humanized antibody against Her 2/neu; and bivatuzumab, which is a humanized antibody against CD44v 6.

In continuing improvements in methods for treating cancer patients, it would be important to develop additional cytotoxic conjugates that specifically recognize specific types of cancer cells.

To this end, the present invention relates to the development of antibodies that recognize and bind to molecules/receptors expressed on the surface of cancer cells, and to the development of novel cytotoxic conjugates comprising a cell-binding agent, such as an antibody, and a cytotoxic agent that specifically targets the molecules/receptors expressed on the surface of cancer cells.

More specifically, the invention relates to the characterization of a novel CA6 sialylsugar epitope (sialoglycotope) located on the Muc1 mucin receptor expressed by cancer cells, to the preparation of antibodies, preferably humanized antibodies, that recognize the novel CA6 sialylsugar epitope of Muc1 mucin and that can be used to inhibit the growth of cells expressing the CA6 sugar epitope in the presence of cytotoxic agents.

Summary of The Invention

The present invention includes antibodies, or epitope-binding fragments thereof, that specifically recognize and bind to a novel CA6 sialylsugar epitope (sialoglycotopope) of the Muc1 mucin receptor. In another embodiment, the invention includes a humanized antibody or epitope-binding fragment thereof that recognizes a novel CA6 sialyl sugar epitope ("CA 6 sugar epitope") of Muc1 mucin receptor.

In a preferred embodiment, the invention encompasses surface reconstituted or humanized forms of the murine anti-CA 6 monoclonal antibody DS6 ("DS 6 antibody") and DS6 antibody, wherein surface exposed residues of the antibody or epitope-binding fragment thereof are substituted in the light and heavy chains to more closely approximate the surface of a known human antibody. The humanized antibodies and epitope-binding fragments thereof of the present invention have improved properties because they are less immunogenic (or completely non-immunogenic) in human subjects to which they are administered than in a fully murine form. Thus, the humanized DS6 antibodies and epitope-binding fragments thereof of the present invention specifically recognize a novel sialic acid sugar epitope on Mucl mucin receptor, i.e., the CA6 sugar epitope, while being non-immunogenic in humans. Humanized antibodies and epitope-binding fragments thereof can be conjugated to drugs such as maytansinoids (maytansinoids) by targeting the drug to the Mucl CA6 sialyl epitope to form pro-drugs (produgs) that are specifically cytotoxic to antigen expressing cells. Cytotoxic conjugates comprising such antibodies and small, highly toxic drugs, such as maytansinoids, taxanes (taxanes), and CC-1065 analogs, can therefore be used as therapeutic agents for the treatment of tumors, such as breast and ovarian tumors.

The humanized forms of the DS6 antibody of the invention are fully characterized herein in terms of: the respective amino acid sequences of the light and heavy chain variable regions, the DNA sequences of the light and heavy chain variable region genes, the identification of the CDRs, the identification of their surface amino acids, and the disclosure of methods for their expression in recombinant form.

In one embodiment, a humanized DS6 antibody or epitope-binding fragment thereof is provided having a heavy chain comprising CDRs having the amino acid sequences set forth in SEQ ID NOS: 1-3: SYNM H (SEQ ID NO: 1), Y I Y P G N G A T N Y N Q K F K G (SEQ ID NO: 2), GdSV P F A Y (SEQ ID NO: 3), and having a light chain comprising CDRs having the amino acid sequences set forth in SEQ ID NOS: 4-6: s A H S S V S F M H (SEQ ID NO: 4), STS S LS A S (SEQ ID NO: 5), Q Q R S S F P L T (SEQ ID NO: 6)

Also provided are humanized DS6 antibodies and epitope-binding fragments thereof, having a light chain variable region having an amino acid sequence that hybridizes to SEQ ID NO: 7 or SEQ ID NO: 8 has at least 90% sequence identity to the amino acid sequence set forth in seq id no: QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFG AGTKLELKR (SEQ ID NO: 7) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSS LASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG AGTKLELKR (SEQ ID NO: 8).

Likewise, humanized DS6 antibodies and epitope-binding fragments thereof are provided that have a heavy chain variable region having an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 9. SEQ ID NO: 10 or SEQ ID NO: 11 has at least 90% sequence identity to the amino acid sequence set forth in seq id no: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 9) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 11).

In another embodiment, humanized DS6 antibodies and epitope-binding fragments thereof are provided having a humanized or resurfaced light chain variable region having an amino acid sequence corresponding to SEQ ID NO: 8 in the amino acid sequence: EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSS LASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG AGTKLELKR (SEQ ID NO: 8).

Likewise, humanized DS6 antibodies and epitope-binding fragments thereof are provided having humanized or resurfaced heavy chain variable regions having amino acid sequences corresponding to SEQ ID NOs: 10 or SEQ ID NO: 11, the amino acid sequence of: QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 11).

The humanized DS6 antibodies and epitope-binding fragments thereof of the present invention may also include substitutions in the light chain and/or heavy chain amino acid residues at one or more positions defined by the asterisked residues in table 1 that represent murine surface framework residues (murine surface framework residues) that are found to be within 5 angstroms of the CDRs and are required to become human residues. For example, the first amino acid residue Q in the murine sequence (SEQ ID NO 7) is replaced by E (SEQ ID NO: 8) to humanize the antibody. However, since this residue is close to the CDR, a back mutation to murine residue Q may be required to maintain antibody affinity. TABLE 1This is further shown in table 2, where the muDS6 variable region surface residues are shown aligned with the three most homologous human variable region surface residues. The amino acid residues in table 1 correspond to the underlined amino acid residues in table 2.

TABLE 2

The invention further provides a cytotoxic conjugate comprising (1) a cell-binding agent that recognizes and binds a CA6 glycoepitope, and (2) a cytotoxic agent. Among cytotoxic conjugates, cell binding agents have high affinity for the CA6 saccharide epitope and cytotoxic agents are highly cytotoxic to cells expressing the CA6 saccharide epitope, so the cytotoxic conjugates of the present invention form effective killing agents.

In a preferred embodiment, the cell binding agent is an anti-CA 6 antibody or epitope-binding fragment thereof, more preferably a humanized anti-CA 6 antibody or epitope-binding fragment thereof, wherein a cytotoxic agent is covalently linked to the antibody or epitope-binding fragment thereof, either directly or through a cleavable or non-cleavable linker. In a more preferred embodiment, the cell binding agent is a humanized DS6 antibody or epitope-binding fragment thereof and the cytotoxic agent is paclitaxel (taxol), a maytansinoid, CC-1065, or a CC-1065 analog.

In a preferred embodiment of the invention, the cell-binding agent is a humanized anti-CA 6 antibody and the cytotoxic agent is a cytotoxic drug, such as a maytansinoid or a taxane (taxane).

More preferably, the cell binding agent is a humanized anti-CA 6 antibody DS6 and the cytotoxic agent is a maytansine compound, e.g., DM1 or DM 4.

The invention also includes methods of inhibiting the growth of cells expressing the carbohydrate epitope of CA 6. In a preferred embodiment, the method for inhibiting the growth of a cell expressing the carbohydrate epitope of CA6 is performed in vivo and results in cell death, although in vitro and ex vivo applications are also included.

The invention also provides a therapeutic composition comprising a cytotoxic conjugate and a pharmaceutically acceptable carrier or excipient.

The invention further includes methods of treating a subject having cancer using the therapeutic compositions. In a preferred embodiment, the cytotoxic conjugate comprises an anti-CA 6 antibody and a cytotoxic agent. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized DS6 antibody-DM 1 conjugate, a humanized DS6 antibody-DM 4 or a humanized DS6 antibody-taxane conjugate, which is administered with a pharmaceutically acceptable carrier or excipient.

The invention also includes kits comprising an anti-CA 6 antibody-cytotoxic agent conjugate and instructions for use. In a preferred embodiment, the anti-CA 6 antibody is a humanized DS6 antibody and the cytotoxic agent is a maytansine compound, such as DM1 or DM4, or a taxane, and the instructions relate to the use of the conjugate to treat a subject having cancer. The kit also includes the components necessary for preparing a pharmaceutically acceptable formulation, such as a diluent if the conjugate is in a lyophilized state or concentrated form, and includes the components for administering the formulation.

The invention also includes derivatives of antibodies that specifically bind to and recognize the carbohydrate epitope of CA 6. In a preferred embodiment, the antibody derivative is prepared by resurfacing or humanizing an antibody that binds to the carbohydrate epitope of CA6, wherein the derivative has reduced immunogenicity to the host.

The invention further provides humanized antibodies or fragments thereof, which are further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion.

Also provided are methods for diagnosis, wherein the labeled humanized antibody or epitope-binding fragment thereof is administered to a receptor suspected of having cancer, and the distribution of the label in the subject is measured or monitored.

The invention also provides methods for treating a subject having cancer by administering the humanized antibody conjugates of the invention, either alone or in combination with other cytotoxic or therapeutic agents. The cancer may be, for example, one or more of breast cancer, colon cancer, ovarian cancer, endometrial cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, a sarcoma or tumor in which CA6 is expressed, or other cancer to be identified in which the CA6 carbohydrate epitope is significantly expressed.

All references and patents cited herein are incorporated by reference unless otherwise indicated.

Drawings

FIG. 1 shows the results of a study to determine the ability of the DS6 antibody to bind to the surface of selected cancer cell lines. Fluorescence of cell lines incubated with DS6 primary antibody and FITC-conjugated anti-mouse IgG (H + L) secondary antibody was measured by flow cytometry. DS6 antibody bound to Caov-3 (FIG. 1A) and T-47D (FIG. 1B) cells with apparent Kd of 1.848nM and 2.586nM, respectively. Antigen-negative cell lines, SK-OV-3 (FIG. 1C) and Colo205 (FIG. 1D) showed no antigen-specific binding.

FIG. 2 shows the results of dot blot analysis of epitope expression. Caov-3 (FIG. 2A & FIG. 2B), SKMEL28 (FIG. 2C) and Colo205 (FIG. 2D) cell lysates were spotted onto nitrocellulose membranes, respectively, followed by incubation with pronase (pronase), proteinase K, neuraminidase or periodic acid. The membrane was then immunoblotted with DS6 antibody (fig. 2A), CM1 antibody (fig. 2B), R24 antibody (fig. 2C) or C242 antibody (fig. 2D).

FIG. 3 shows the results of dot blot analysis of DS6 antigen expression. Caov-3 cell lysates were each spotted onto PVDF membranes and then incubated in the presence of trifluoromethanesulfonic acid (TFMSA). The membrane was then immunoblotted with either CM1 antibody (1&2) or DS6 antibody (3& 4).

FIG. 4 shows the results of carbohydrate epitope analysis of the DS6 antigen. Caov-3 lysates pretreated with N-glycanase ("N-gly"), O-glycanase ("O-gly"), and/or sialidase ("S") were spotted onto nitrocellulose, followed by immunoblotting with either DS6 antibody or CM1 antibody (Muc-1 VNTR).

Fig. 5 shows the results of western blot analysis of DS6 antigen. Cell lysates were immunoprecipitated ("IP") and immunoblotted with DS6 antibody. The antigen corresponded to a protein band of greater than 250kDa, as observed in antigen-positive Caov-3 (FIGS. 5A and 5B) and T47D (FIG. 5C) cells. Antigen-negative SK-OV-3 (fig. 5D) and Colo205 (fig. 5E) cell lines did not show this band. Following the immunoprecipitation, the protein G beads of the Caov-3 cell lysate were incubated with either neuraminidase ("N") (FIG. 5A) or periodic acid ("PA") (FIG. 5B). Antibody ("α"), pre-IP ("Lys") and post-IP flow through ("FT") lysate controls were analyzed on the same gel. The Caov-3 immunoprecipitates were also incubated with N-glycanase ("N-gly"), O-glycanase ("O-gly") and/or sialidase ("S") (see FIG. 5F) where the blots were probed with biotinylated-DS 6 and streptavidin-HRP.

FIG. 6 shows the results of immunoprecipitation reactions and/or immunoblots of DS6 antibody and CM1 antibody on Caov-3 (FIG. 6A) and HeLa (FIG. 6B) cell lysates. The addition of CM1 and DS6 Western blot signals indicated that the DS6 antigen is on the Muc1 protein. In the HeLa lysate, the Muc1 double product results from the expression of Muc1, directed by different alleles with different numbers of tandem repeats.

Figure 7 shows the DS6 antibody sandwich ELISA design (figure 7A) and the standard curve (figure 7B). A standard curve was generated using a known concentration of a commercially available CA15-3 standard (where 1CA 15-3 units ═ 1 DS6 units).

Figure 8 shows a quantitative ELISA standard curve. The standard curve of the signal of the detection antibody (streptavidin-HRP/biotin-DS 6) was determined (fig. 8C) using a known concentration of biotin-DS 6, either captured by goat anti-mouse IgG on the plate (fig. 8A) or bound directly to an ELISA plate (fig. 8B).

FIG. 9 shows the cDNA and amino acid sequences of the light chain (FIG. 9A) and heavy chain (FIG. 9B) variable regions of murine DS6 antibody. Three CDRs in each sequence are underlined (Kabat definition).

Figure 10 shows the light chain (figure 10A) and heavy chain (figure 10B) CDRs of a murine DS6 antibody, as determined by Kabat definition. For the heavy chain CDRs, the AbM modeling software yielded slightly different definitions (fig. 10C). [44] FIG. 11 shows the light chain ("mudS 6 LC") (residues 1-95 of SEQ ID NO: 7) and heavy chain ("mudS 6 HC") (residues 1-98 of SEQ ID NO: 9) amino acid sequences of murine DS6 antibody aligned (align) with germline sequences of the IgVap4(SEQ ID NO: 23) and IgVh J558.41(SEQ ID NO: 24) genes. Sequence differences are indicated in grey.

FIG. 12 shows the 10 light and heavy chain antibody sequences with resolved structure (solved structure) files in the Brookhaven database that are most homologous to the murine DS6(mudS6) light chain ("mudS 6 LC") and heavy chain ("mudS 6 HC") sequences. The sequences are arranged in order of most to least homology.

Fig. 13 shows surface accessibility data and calculations used to predict which framework residues (framework residues) of the murine DS6 antibody light chain variable region are surface accessible. Locations with average surface accessibility of 25-35% are marked ('^*) They are subjected to a second round of analysis. The DS6 antibody light chain variable region (fig. 13A) and heavy chain variable region (fig. 13B).

Fig. 14 shows a prDS 6v1.0 mammalian expression plasmid map. This plasmid was used to establish and express recombinant chimeric and humanized DS6 antibodies.

FIG. 15 shows the amino acid sequences of the light chain (FIG. 15A) and heavy chain (FIG. 15B) variable domains of murine ("mudS 6") and humanized ("huDS 6") (v1.0& v1.2) DS6 antibodies.

FIG. 16 shows the cDNA and amino acid sequences of the light chain variable region of the humanized DS6 antibody ("huDS 6") (v1.0 and v 1.2).

Figure 17 shows the cDNA and amino acid sequences of the heavy chain variable regions of humanized DS6 antibodies v1.0 (figure 17A) and v1.2 (figure 17B).

FIG. 18 shows flow cytometry binding curves for mudS6 and huDS clones from assays performed on WISH cells. (fig. 18A) the binding affinities (Kd ═ 3.15, 3.71 and 4.20nM) of the chimeric, v1.0 and v1.2 human DS6 clones were compared to (fig. 18B) naked DS6 and biotinylated murine DS6(Kd ═ 1.93 and 2.80 nM).

FIG. 19 shows the results of a competitive binding assay of huDS6 antibody to mudS 6. (FIG. 19A) incubation of WISH cells with biotin-mudS 6 and streptavidin-DTAF produced a binding curve with an apparent Kd of 6.76 nM. (FIG. 19B) varying concentrations of naked mudS6, huDS6v1.0, and v1.2 were mixed with 2nM secondary reagents of biotin-mudS 6 and streptavidin-DTAF.

Fig. 20 shows the results of the binding affinity assay for the unconjugated DS6 antibody and the DS6 antibody-DM 1 conjugate. The results show that DM1 conjugation did not adversely affect the binding affinity of the antibody. The apparent Kd (3.902nM) of the DS6 antibody-DM 1 conjugate ("DS 6-DM 1") was slightly greater than that of the naked antibody (2.020nM) ("DS 6").

Figure 21 shows the results of an indirect cell viability assay using the DS6 antibody in the presence or absence of an anti-mouse IgG (H + L) DM1 conjugate (2 ° Ab-DM 1). Antigen-positive Caov-3 cells (IC) were killed in a DS6 antibody-dependent manner only in the presence of a secondary conjugate ("DS 6+2 ° Ab-DM1₅₀＝424.9pM)。

Fig. 22 shows the results of Complement Dependent Cytotoxicity (CDC) assays for DS6 antibody and humanized DS6 antibody. The results showed that there was no CDC mediated effect (mediated effect) of DS6 antibody or humanized DS6 antibody on HPAC (FIG. 22A) and ZR-75-1 (FIG. 22B) cells.

FIG. 23 shows the results of in vitro cytotoxicity assays of DS6 antibody-DM 1 conjugate and free maytansine. In a colony-generating assay (clonogenic assay), DS6 antigen-positive ovarian (fig. 23A), breast (fig. 23B), cervical (fig. 23C), and pancreatic (fig. 23D) cancer cell lines were serially exposed to DS6 antibody-DM 1 conjugate, and tested for cytotoxicity (left panel). Maytansine sensitivity of these cell lines was similarly determined by 72h exposure to free maytansine (right panel). Ovarian cancer cell lines tested were OVCAR5, TOV-21G, Caov-4, and Caov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483. The cervical cancer cell lines tested were KB, HeLa and WISH. The pancreatic cancer cell lines tested were HPAC, Hs766T and HPAF-11.

FIG. 24 shows the results of in vitro cytotoxicity assays of DS6 antibody-DM 1 conjugate. In the MTT cell viability assay, human ovarian (fig. 24A, fig. 24B & fig. 24C), breast (fig. 24D & fig. 24E), cervical (fig. 24F & fig. 24G), and pancreatic (fig. 24H & fig. 24I) cancer cells were killed in a manner dependent on the DS6 antibody-DM 1 conjugate. Naked DS6 did not adversely affect the growth of these cells, indicating that DM1 conjugation is required for cytotoxicity.

Figure 25A shows the results of an in vivo anti-tumor efficacy study of DS6 antibody-DM 1 conjugate on an established subcutaneous KB tumor xenograft. Tumor cells were inoculated on day 0 and the first treatment was given on day 6. The immunoconjugate treatment was performed continuously daily for a total of 5 doses. Once the tumor volume exceeded 1500mm³PBS control animals were euthanized. The conjugate was administered at a dose of 150 or 225 μ g/kg DM1, corresponding to antibody concentrations of 5.7 and 8.5mg/kg, respectively. Body weight of the mice was monitored during the study (fig. 25B).

Figure 26 shows the results of an in vivo anti-tumor efficacy study of DS6 antibody-DM 1 conjugate on an established subcutaneous tumor xenograft. OVCAR5 (FIGS. 26A and 26B), TOV-21G (FIGS. 26C and 26D), HPAC (FIGS. 26E and 26F), and HeLa (FIGS. 26G and 26H) cells were seeded at day 0 and immunoconjugates were administered on days 6 and 13. Once the tumor volume exceeds 1000mm³PBS control animals were euthanized. The conjugate was administered at a dose of 600. mu.g/kg DM1, corresponding to an antibody concentration of 27.7 mg/kg. Tumor volume (fig. 26A, 26C, 26E and 26G) and body weight (fig. 26B, 26D, 26F and 26H) were monitored in mice during the study.

Figure 27 shows the results of in vivo efficacy studies of DS6 antibody-DM 1 conjugate on intraperitoneal OVCAR5 tumors. Tumor cells were injected intraperitoneally on day 0 and immunoconjugate treatments were given on days 6 and 13. Once the weight loss exceeded 20%, the animals were euthanized.

Figure 28 shows flow cytometry binding curves for binding affinity studies of naked DS6 and taxane-conjugated DS6 antibodies on HeLa cells. Taxane (MM1-202) conjugation did not adversely affect the binding affinity of the antibody. The apparent Kd (1.24nM) of the DS6-MM1-202 conjugate was slightly greater than that of the naked DS6 antibody (620 pM).

Detailed Description

The present invention provides fragments of anti-CA 6 monoclonal antibodies, anti-CA 6 humanized antibodies, and anti-CA 6 antibodies. Each of the antibodies and antibody fragments of the invention are designed to specifically recognize and bind to the CA6 glycoepitope on the cell surface. CA6 is known to be expressed by many human tumors: 95% serous ovarian cancer (serous ovarcorcinogens), 50% endometrial ovarian cancer (endometeroid ovarcomas), 50% cervical tumors (neoplasms of the endometrial cervix), 69% endometrial tumors (neoplasms of the endometrioid), 80% vulvar tumors (neoplasms of the vulva), 60% breast cancer, 67% pancreatic tumors, and 48% urothelium tumors (tumors of the urothelium), but it is rarely expressed by normal human tissues.

Cancer 88(6) in Kearse et al, int.j: 866-872(2000), when they used the hybridoma supernatant to identify the protein on which the CA6 epitope was found, they incorrectly identified it as an 80 kDa protein with the N-linked sugar containing the CA6 epitope. Using purified DS6, we have demonstrated that the CA6 epitope is found on the O-linked sugar of a non-disulfide linked glycoprotein of greater than 250 kDa. In addition, the glycoprotein was identified as mucin, Muc 1. Because different Muc1 alleles have different numbers of tandem repeats in an indeterminate number of tandem repeat (VNTR) domains, cells often express two different Muc1 proteins with different sizes (Taylor-Papademitou, Biochim.Biophys.acta 1455 (2-3): 301-13 (1999). due to the different numbers of repeats in the VNTR domains and differences in glycosylation, the molecular weight of Muc1 varies from cell line to cell line.

The sensitivity of the immunoreactivity of CA6 to periodic acid indicated that CA6 is the carbohydrate epitope, "glycoepitope". Additional sensitivity of CA6 immunoreactivity to treatment with neuraminidase from Vibrio cholerae (Vibrio cholerae) suggests that the CA6 epitope is a sialic acid-dependent sugar epitope and thus a "sialoglycotopoe".

Characterization details of CA6 can be found in example 2 (see below). May be found in WO 02/16401; wennerberg et al, am.J. Patlzol.143 (4): 1050-1054 (1993); smith et al, Human Antibodies 9: 61-65 (1999); kearse et al, int.j. cancer 88 (6): 866-872 (2000); smith et al, int.JGynecol.Pathol.20 (3): 260-6(2001) and Smith et al, appl.immunohistochem.mol.morphol.10 (2): 152-8(2002) find additional details regarding CA 6.

The invention also includes cytotoxic conjugates comprising two major components. The first component is a cell-binding agent that recognizes and binds the CA6 carbohydrate epitope. The cell binding agent should recognize the CA6 sialyl epitope on Muc1 with a high degree of specificity, so the cytotoxic conjugate only recognizes and binds the desired cell. The high degree of specificity allows the conjugate to function in a targeted manner with little side effects caused by non-specific binding.

In another embodiment, the cell binding agent of the invention also recognizes the CA6 glycoepitope with high affinity, and thus the conjugate will be in contact with the target cell for a sufficient time such that the cytotoxic drug moiety of the conjugate can act on the cell and/or such that there is sufficient time for the cytotoxic drug moiety to be internalized by the cell.

In a preferred embodiment, the cytotoxic conjugate comprises an anti-CA 6 antibody as a cell binding agent, more preferably a murine DS6 anti-CA 6 monoclonal antibody. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized DS6 antibody or epitope-binding fragment thereof. The DS6 antibody recognizes CA6 with high specificity, directing cytotoxic agents to abnormal cells or tissues, such as cancer cells, in a targeted manner.

Another component of the cell conjugates of the invention is a cytotoxic agent. In a preferred embodiment, the cytotoxic agent is paclitaxel, a maytansinoid such as DM1 or DM4, CC-1065, or a CC-1065 analog. In a preferred embodiment, the cell binding agent of the invention is covalently linked to a cytotoxic agent, either directly or via a cleavable or non-cleavable linker (linker).

Cell binding agents, cytotoxic agents, and linkers are discussed in more detail below.

Cell binding agents

The effectiveness of the compounds of the invention as therapeutic agents depends on the careful selection of an appropriate cell-binding agent. The cell binding agent may be of any kind currently known, or to be known, and includes peptides and non-peptides. The cell-binding agent can be any compound that can bind to a cell in a specific or non-specific manner. In general, these agents may be antibodies (particularly monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport factors (e.g., transferrin), or any other cell binding molecule or substance.

More specific examples of cell-binding agents that may be used include:

(a) a polyclonal antibody;

(b) a monoclonal antibody;

(c) antibody fragments, e.g. Fab, Fab 'and F (ab')₂Fv (Parham, J.Immunol.131: 2895-2902 (1983); Spring et al, J Immunol.113: 470-478 (1974); Nisonoff et al, Arch.biochem.Biophys.89: 230-244 (1960));

(d) interferons (e.g., α, β, γ);

(e) lymphokines such as IL-2, IL-3, IL-4, IL-6;

(f) hormones, such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones, such as androgens and estrogens;

(g) growth factors and colony stimulating factors, such as EGF, TGF- α, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984));

(h) transferrin (O' Keefe et al, J.biol.chem.260: 932-937 (1985)); and

(i) vitamins, such as folic acid.

Antibodies

Selection of an appropriate cell-binding agent is critical to the selection, depending on the particular cell population to be targeted, but in general, antibodies are preferred, more preferably monoclonal antibodies, if an appropriate antibody can be utilized or can be prepared.

Monoclonal antibody technology allows the production of very specific cell-binding agents in the form of specific monoclonal antibodies. The technology of making monoclonal antibodies by immunizing mice, rats, hamsters or any other mammal with an antigen of interest, e.g., intact target cells, antigens isolated from target cells, intact viruses, attenuated intact viruses and viral proteins such as viral capsid proteins, is well known in the art. Sensitized human cells may also be used. Another method of making monoclonal antibodies is to use phage libraries of scFv (single chain variable regions), especially human scFv (see, e.g., Griffiths et al, U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al, WO 92/01047; Liming et al, WO 99/06587).

A typical antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. The variable region is a portion of the heavy and light chains of an antibody that has different sequences within the antibody and which incorporates the binding and specificity of each particular antibody for its antigen. Variability is not usually evenly distributed throughout the antibody variable region. It is typically concentrated within three segments of the variable region, called Complementarity Determining Regions (CDRs) or hypervariable regions, located within the light and heavy chain variable regions. The portion of the variable region with higher conservation is called the framework region. The variable regions of the heavy and light chains comprise four framework regions, predominantly in the β -sheet configuration, in which the framework regions are connected by three CDRs which form a loop structure connecting, and in some cases forming part of, the β -sheet structure. The CDRs in each chain are held in positional proximity by the framework regions and together with the CDRs from the other chain help form the antigen binding site of the antibody (e.a. kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, 1991, NIH).

The constant region is part of the heavy chain. Although not directly involved in binding an antibody to an antigen, it does exhibit various effector functions (effects), such as participation of an antibody in antibody-dependent cytotoxicity.

Suitable monoclonal antibodies for use in the present invention include the murine DS6 monoclonal antibody (U.S. Pat. No. 6,596,503; ATCC accession number PTA-4449).

Humanized or resurfaced DS6 antibodies

Preferably, a humanized anti-CA 6 antibody is used as the cell binding agent of the invention. A preferred embodiment of such a humanized antibody is a humanized DS6 antibody, or epitope-binding fragment thereof.

The goal of humanization is to reduce the immunogenicity of xenogenous antibodies, such as murine antibodies, introduced into the human body, while maintaining the intact antigen-binding affinity and specificity of the antibody.

Humanized antibodies can be generated using several techniques, such as resurfacing (resurfacing) and CDR grafting (grafting). As used herein, surface reconstruction techniques use a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surfaces of antibody variable regions to mimic the surface of known antibodies of a target host.

Strategies and methods for the resurfacing of antibodies, as well as other methods for reducing the immunogenicity of antibodies in different hosts, are disclosed in U.S. Pat. No. 5,639,641(Pedersen et al), the entire contents of which are incorporated herein by reference. Briefly, in a preferred method, (1) a series of antibody heavy chains are produced andaligning (alignments) the positions of the light chain variable regions to provide a set of positions exposed at the surface of the heavy and light chain variable region frameworks, wherein the aligned positions of all variable regions are at least about 98% identical; (2) defining for the rodent antibody (or fragment thereof) a set of amino acid residues exposed at the surface of the heavy and light chain variable region frameworks; (3) identifying a set of surface exposed amino acid residues in the heavy and light chain variable region frameworks that are most closely identical to the set of rodent surface exposed amino acid residues; (4) substituting the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3) for the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2), except 5 of any atom of any residue located in the complementarity determining region of the rodent antibodyAmino acid residues within; and (5) generating a humanized rodent antibody with binding specificity.

Antibodies can be humanized using a variety of other techniques, including CDR-grafting (EP 0239400; WO 91/09967; U.S. Pat. No. 5,530,101; and 5,585,089), veneering (surfacing) or resurfacing (EP 0592106; EP 0519596; Padlan E.A., 1991, Molecular Immunology 28 (4/5): 489-498; Studnica G.M. et al, 1994, Protein Engineering 7 (6): 805-814; Roguska M.A. et al, 1994, PNAS 91: 969-973) and chain rearrangement (U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety of techniques known in the art, including phage display methods. See U.S. Pat. nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International patent application publication Nos. WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (the entire contents of which are incorporated herein by reference).

In a preferred embodiment, the present invention provides a humanized antibody or fragment thereof that recognizes a novel sialic acid sugar epitope (CA6 sugar epitope) on Muc1 mucin. In another embodiment, the humanized antibody or epitope-binding fragment thereof has the additional ability to inhibit the growth of cells expressing the carbohydrate epitope of CA 6.

In a more preferred embodiment, a resurfaced or humanized form of the DS6 antibody is provided in which surface exposed residues of the antibody or fragment thereof are replaced in the light and heavy chains to more closely resemble known human antibody surfaces. The humanized DS6 antibodies or epitope-binding fragments thereof of the invention have improved properties. For example, the humanized DS6 antibody or epitope-binding fragment thereof specifically recognizes a novel sialic acid sugar epitope (CA6 sugar epitope) on Muc1 mucin. More preferably, the humanized DS6 antibody or epitope-binding fragment thereof has the additional ability to inhibit the growth of cells expressing the CA6 carbohydrate epitope. The humanized antibody or epitope-binding fragment thereof can be linked to a drug such as a maytansinoid by targeting the drug to the novel Muc1 sialyl epitope, CA6, to form a prodrug that is specifically cytotoxic to antigen expressing cells. Cytotoxic conjugates comprising such antibodies and small, highly toxic drugs (e.g., maytansinoids, taxanes, and CC-1065 analogs) may be used as therapeutic agents for the treatment of tumors such as and ovarian cancer.

Humanized forms of the DS6 antibody are also fully characterized herein and methods for their respective amino acid sequences of the heavy and light chain variable regions, DNA sequences of the light and heavy chain variable region genes, identification of the CDRs, identification of surface amino acids thereof, and their expression in recombinant form are disclosed.

In one embodiment, a humanized antibody or epitope-binding fragment thereof is provided having a heavy chain comprising CDRs having a sequence consisting of SEQ ID NOs: 1-3:

S Y N M H(SEQ ID NO：1)

Y I Y P G N G A T N Y N Q K F K G(SEQ ID NO：2)

G D S V P F A Y(SEQ ID NO：3)

when the heavy chain CDRs were determined by AbM modeling software, they were determined from SEQ ID NOs: 20-22 represent:

G Y T F T S Y N M H(SEQ ID NO：20)

Y I Y P G N G A T N(SEQ ID NO：21)

G D S V P F A Y(SEQ ID NO：22)

in the same embodiment, the humanized antibody or epitope-binding fragment thereof has a light chain comprising CDRs having the amino acid sequences set forth in SEQ ID NOS: 4-6:

S A H S S V S F M H(SEQ ID NO：4)

S T S S L A S(SEQ ID NO：5)

Q Q R S S F P L T(SEQ ID NO：6)

also provided are humanized antibodies and epitope-binding fragments thereof having a light chain variable region with an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 share at least 90% sequence identity:

QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSS

LASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFG

AGTKLELKR(SEQ ID NO：7)

EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSS

LASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG AGTKLELKR(SEQ ID NO：8)。

similarly, humanized antibodies and epitope-binding fragments thereof are provided having a heavy chain variable region with an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 10 or SEQ ID NO: 11 share at least 90% sequence identity: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 9) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 11).

In another embodiment, humanized antibodies and epitope-binding fragments thereof are provided having a humanized or resurfaced light chain variable region having an amino acid sequence corresponding to SEQ ID NO: 8; EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSS LASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG AGTKLELKR (SEQ ID NO: 8).

Likewise, humanized antibodies and epitope-binding fragments thereof are provided having a humanized or resurfaced heavy chain variable region having an amino acid sequence corresponding to SEQ ID NO: 10 or SEQ ID NO: 11, the amino acid sequence of: QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 11).

The humanized antibodies and epitope-binding fragments thereof of the present invention may also include the form of light and/or heavy chain variable regions in which the human surface amino acid residues in close proximity to the CDRs are substituted with the corresponding surface residues of muDS6 at one or more positions defined by the asterisked residues in table 1(Kabat numbering) in order to preserve the binding affinity and specificity of muDS 6. TABLE 1

Disclosed herein are the major amino acid and DNA sequences of the light and heavy chains of the DS6 antibody, and the major amino acid and DNA sequences of humanized versions thereof. However, the scope of the invention is not limited to antibodies and fragments comprising these sequences. Rather, all antibodies and fragments that specifically bind CA6 as a distinct tumor-specific carbohydrate epitope on the Muc1 receptor are included in the present invention. Preferably, antibodies and fragments that specifically bind CA6 also antagonize the biological activity of the receptor. More preferably, such antibodies are further substantially free of agonist activity. Thus, the antibodies and antibody fragments of the invention may differ from the DS6 antibody or humanized derivatives thereof in the amino acid sequence, CDRs and/or light and heavy chains of its framework (scaffold), while still being within the scope of the invention.

The CDRs of the DS6 antibody were identified by modeling and their molecular structure was predicted. Furthermore, although the CDRs are important for epitope recognition, they are not essential for the antibodies and fragments of the invention. Thus, antibodies and fragments with improved properties are provided, which are produced, for example, by affinity maturation (affinity purification) of the antibodies of the invention.

The murine light chain IgVap4 germline gene and the heavy chain IgVh J558.41 germline gene from which DS6 was most likely derived are shown in FIG. 11, aligned with the sequence of the DS6 antibody. This comparison identified possible somatic mutations in the DS6 antibody, including several somatic mutations in the CDRs.

The sequences of the heavy and light chain variable regions of the DS6 antibody and the sequences of the CDRs of the DS6 antibody are not previously known and are illustrated in fig. 9A and 9B. Such information can be used to generate humanized versions of DS6 antibodies.

Antibody fragments

Antibodies of the invention include full length antibodies as well as epitope-binding fragments as discussed above. As used herein, "antibody fragment" includes any portion of an antibody that retains the ability to bind to an epitope recognized by a full-length antibody, and is generally referred to asAn "epitope binding fragment". Examples of antibody fragments include, but are not limited to, Fab 'and F (ab')₂Fd, single chain Fvs (scFv), single chain antibody, disulfide linked Fvs (dsFv) and antibodies comprising V_LOr V_HA segment of a region. Epitope-binding fragments, including single chain antibodies, may comprise the variable region alone or in combination with the entire or partial regions of: hinge region, C_H1、C_H2 and C_H3 domain.

Such fragments may comprise one or two Fab fragments or F (ab')₂And (3) fragment. Preferably, the antibody fragment comprises all six CDRs of the whole antibody, although fragments comprising fewer such regions, e.g., three, four or five CDRs, are also functional. Furthermore, fragments may be or may be combined with any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE and subclasses thereof.

Fab and F (ab')₂Examples of the enzyme used for the fragment include papain (Fab fragment) and pepsin (F (ab')₂Fragments).

Single chain FVs (scFvs) fragments are epitope-binding fragments comprising an antibody light chain variable region (V)_L) And an antibody heavy chain variable region (V) linked thereto_H) At least one fragment. The linker may be a short flexible peptide selected to ensure that once (V)_L) And (V)_H) When the regions are linked, appropriate three-dimensional folding can occur so as to preserve the target molecule binding specificity of the intact antibody from which the single-chain antibody fragment is derived. (V)_L) Or (V)_H) The carboxy terminus of the sequence may be linked to the complement (V) by a linker (linker)_L) Or (V)_H) The amino acid termini of the sequences are covalently linked.

Single chain antibody fragments of the invention comprise amino acid sequences having at least one variable region or Complementarity Determining Region (CDRs) of an intact antibody as described in the specification, but lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major part of the complete antibody structure. Single chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies comprising part or all of the constant domains. For example, single chain antibody fragments often do not have undesirable interactions between biomolecules and heavy chain constant regions, or other undesirable biological activities. In addition, single chain antibody fragments are much smaller than whole antibodies and therefore can have greater capillary permeability than whole antibodies, which allows the single chain antibody fragments to more efficiently localize (localize) and bind to the target antigen binding site. Furthermore, antibody fragments can be produced in prokaryotic cells on a relatively large scale, thus facilitating their production. In addition, the relatively small size of single chain antibody fragments makes them less likely to elicit an immune response in a recipient than an intact antibody.

Single chain antibody fragments may be generated by molecular cloning, antibody phage display libraries, or similar techniques well known to those of ordinary skill. For example, these proteins can be produced in eukaryotic or prokaryotic cells, including bacteria. The epitope-binding fragments of the invention can also be generated using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, such phage can be used to display epitope binding domains expressed by a lineage or combinatorial antibody library (e.g., human or murine). The antigen may be used to select or identify phage that express an epitope binding domain that binds to the antigen of interest, for example using a labeled antigen that is bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage, including fd and M13, with binding domains expressed by the phage, and Fab, Fv or disulfide-stabilized Fv antibody domains fused by recombinant methods to the phage gene III or gene VIII protein.

Examples of phage display methods that can be used to prepare epitope-binding fragments of the invention include those described in Brinkman et al, 1995, j.immunol.methods 182: 41-50; ames et al, 1995, j.immunol.methods 184: 177-186 parts of a base; ketleborough et al, 1994, eur.j.immunol.24: 952-958; persic et al 1997, Gene 187: 9-18; burton et al, 1994, Advances in Immunology 57: 191 to 280 parts; PCT application PCT/GB 91/01134; PCT publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753, respectively; 5,821,047, respectively; 5,571,698; 5,427,908; 5,516,637; 5,780,225, respectively; 5,658,727, respectively; 5,733,743 and 5,969,108, each of which is incorporated herein by reference in its entirety.

Following phage selection, the phage portion encoding the fragment can be isolated and used to produce epitope-binding fragments by expression in a selected host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, using recombinant DNA techniques, for example, as described in detail below. For example, recombinant production of Fab, Fab 'and F (ab')₂Fragment techniques, using methods known in the art, such as those disclosed in PCT publication Nos. WO 92/22324; mullinax et al, 1992, BioTechniques 12 (6): 864-869; sawai et al, 1995, AJRI 34: 26-34 and Better et al, 1988, Science 240: 1041-1043; the entire contents of the above references are incorporated by reference. Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Pat. nos. 4,946,778 and 5,258,498; huston et al, 1991, Methods in Enzymology 203: 46-88; shu et al, 1993, PNAS 90: 7995-7999; skerra et al, 1988, Science 240: 1038-1040.

Functional equivalents

Functional equivalents of the anti-CA 6 antibody and the humanized anti-CA 6 antibody are also included within the scope of the present invention. For example, the term "functional equivalents" includes antibodies, chimeric antibodies, artificial antibodies, and modified antibodies having homologous sequences, wherein each functional equivalent is defined as having the ability to bind CA 6. The skilled artisan will appreciate that there is overlap in the population of molecules referred to as "antibody fragments" and the population referred to as "functional equivalents". Methods for producing functional equivalents are described, for example, in PCT application WO 93/21319, European patent application 239,400; PCT application WO 89/09622; european patent application 338,745 and european patent application EP 332,424, the entire contents of which are incorporated by reference.

Antibodies having homologous sequences are those having amino acid sequences having sequence homology to the amino acid sequences of the anti-CA 6 antibody and humanized anti-CA 6 antibody of the invention. Preferably, homology is with the amino acid sequence homology of the variable regions of the anti-CA 6 antibody and humanized anti-CA 6 antibody of the invention. "sequence homology" as applied herein to amino acid sequences is defined as at least about 90%, 91%, 92%, 93% or 94% sequence homology, more preferably at least about 95%, 96%, 97%, 98% or 99% sequence homology of one sequence to another, for example, as determined by the FASTA search method in Pearsonand Lipman, Proc.Natl.Acad.Sci.USA 85,2444-2448 (1988).

As used herein, a chimeric antibody is an antibody in which different portions of the antibody are derived from different animal species. For example, antibodies having variable regions derived from murine monoclonal antibodies are paired with human immunoglobulin constant regions. Methods of producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229: 1202; oi et al, 1986, BioTechniques 4: 214; gillies et al, 1989, j. immunol. methods 125: 191-202; us patent 5,807,715; 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.

Humanized forms of chimeric antibodies are prepared by replacing complementarity determining regions of, for example, a murine antibody into a human framework domain, see, for example, PCT publication No. WO 92/22653. Humanized chimeric antibodies preferably have constant and variable regions, but do not have Complementarity Determining Regions (CDRs) that are substantially or exclusively derived from regions of a corresponding human antibody, but rather have CDRs that are substantially or exclusively derived from a non-human mammal.

Artificial antibodies include scFv fragments, diabodies, triabodies, tetrabodies and mru (see review Winter, G.and Milstein, C., 1991, Nature 349: 293-299; Hudson, P.J., 1999, Current Opinion in Immunology 11: 548-557), each of which has antigen binding ability. In single chain Fv fragments (scFv), V of the antibody_HAnd V_LThe domains are linked by flexible peptides. Typically, the linker peptide is about 15 amino acid sequences in length. If the linker is smaller, e.g., 5 amino acids, a dimeric antibody is formed, which is a bivalent scFv dimer. If the linker is reduced to less than three amino acid residues, trimeric and tetrameric structures called trimeric and tetrameric antibodies are formed. The smallest binding unit of an antibody is the CDR, typically CDR2 of the heavy chain, which has specific recognition and binding capabilities and therefore can be used alone. Such fragments are called molecular recognition units (molecular recognition units) or mru. Several such mrus can be linked together with short linker peptides to form an artificial binding protein with higher affinity (avidity) than a single mru.

Functional equivalents of the present application also include modified antibodies, e.g., antibodies modified by covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies that are modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins. Covalent attachment does not prevent the antibody from producing an anti-idiotypic response. These modifications can be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the modified antibody may comprise one or more non-canonical amino acids (non-canonical amino acids).

Functional equivalents can be generated by exchanging different CDRs in different frameworks on different chains. Thus, for example, by substitution of different heavy chains, different classes of antibodies are possible for a particular set of CDRs, e.g., IgG1-4, IgM, IgA1-2, IgD, IgE antibody classes and isoforms may thereby be produced. Likewise, artificial antibodies included within the scope of the invention may be generated by embedding a particular set of CDRs within a completely synthetic framework.

Functional equivalents can be readily generated by mutating, deleting and/or inserting within the variable and/or constant region sequences flanking a particular set of CDRs using a number of methods known in the art.

Antibody fragments and functional equivalents of the present invention include those molecules that bind to CA6 to a detectable degree as compared to the DS6 antibody. Detectable degrees of binding include all values within the range of at least 10% to 100%, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of the binding capacity of the murine DS6 antibody to CA 6.

Improved antibodies

The CDRs are important for epitope recognition and antibody binding. However, changes can be made to the residues that make up the CDRs without interfering with the ability of the antibody to recognize and bind its cognate epitope. For example, changes can be made that do not affect epitope recognition, but increase the binding affinity of the antibody to the epitope.

Thus, improved forms of murine and humanized antibodies are also included within the scope of the invention, which also specifically recognize and bind CA6, preferably with increased affinity.

Several studies have investigated the effect of introducing one or more amino acid changes at various positions in the antibody sequence on its properties, such as binding and expression levels, based on knowledge of the original antibody sequence (Yang, W.P. et al, 1995, J.mol.biol., 254, 392-403; Rader, C.et al, 1998, Proc.Natl.Acad.Sci.USA, 95, 8910-8915; Vaughan, T.J. et al, 1998, Nature Biotechnology 539, 16, 535-539).

In these studies, the equivalent of the naive antibody has been generated by altering the heavy and light chain gene sequences in the CDR1, CDR2, CDR3 or the framework regions using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone polymerase chain reaction, DNA rearrangement or mutator strains of E.coli (Vaughan, T.J. et al, 1998, Nature Biotechnology, 16, 535-539; Adey, N.B. et al, 1996, Chapter 16, pp.277-291, in "Phage Display of Peptides and Proteins", eds. Kay, B.K. et al, academic Press). These methods of altering the sequence of the primary antibody have resulted in improved affinity for the secondary antibody (Gram, h. et al, 1992, proc. natl. acad. sci. usa, 89, 3576-3580; Boder, e.t. et al, 2000, proc. natl. acad. sci. usa, 97, 10701-10705; Davies, j.and Riechmann, l. 1996, immunotechnology, 2, 169-179; Thompson, j. et al, 1996, j. mol. biol. 256, 77-88; Short, m.k. et al, 2002, j.biol. chem. 277, 16365-16370; Furukawa, k. et al, 2001, j.276. chem. 622, 27628).

By such similar targeting strategies as altering one or more amino acid residues of the antibody, the antibody sequences described in the present invention can be used to develop anti-CA 6 antibodies with improved function, including improved affinity for CA 6.

Improved antibodies also include those with improved characteristics, which are prepared by standard techniques of animal immunization, hybridoma formation, and selection of antibodies with specific characteristics.

Cytotoxic agents

The cytotoxic agent used in the cytotoxic conjugate of the invention may be any compound that causes or induces cell death or in some way reduces cell viability. For example, preferred cytotoxic agents include maytansinoids and maytansinoid analogs, taxanes (taxoids), CC-1065 and CC-1065 analogs, dolastatins (dolastatins), and dolastatin analogs, as defined below. These cytotoxic agents are conjugated to the antibodies, antibody fragments, functional equivalents, improved antibodies, and analogs thereof disclosed herein.

Cytotoxic conjugates can be prepared by in vitro methods. For linking the drug or prodrug and the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Preferred linking groups are disulfide groups and thioether groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between the antibody and the drug or prodrug.

Maytansinoids

Maytansinoids and maytansinoid analogs are among the cytotoxic agents that may be used in the present invention to form cytotoxic conjugates. Examples of suitable maytansinoids include maytansinol (maytansinol) and maytansinol analogs. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

Examples of suitable maytansinol analogues include those having modified aromatic rings and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. patent 4,424,219; 4,256,746, respectively; 4,294,757, respectively; 4,307,016, respectively; 4,313,946, respectively; 4,315,929, respectively; 4,331,598, respectively; 4,361,650, respectively; 4,362,663, respectively; 4,364,866, respectively; 4,450,254, respectively; 4,322,348, respectively; 4,371,533, respectively; 6,333,410; 5,475,092; 5,585,499 and 5,846,545.

Specific examples of suitable maytansinol analogues having a modified aromatic ring include:

(1) c-19-dechlorination (U.S. Pat. No. 4,256,746) (prepared by LAH reduction of ansamytocin P2);

(2) c-20-hydroxy (or C-20-demethyl) +/-C-19-dechlorinated (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces (Streptomyces) or Actinomycetes (Actinomyces) or by dechlorination using LAH); and

(3) c-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechlorination (U.S. Pat. No. 4,294,757) (prepared by acylation using an acid chloride).

Specific examples of suitable maytansinol analogues with modifications at other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (by reaction of maytansinol with H₂S or P₂S₅By reaction of (a);

(2) C-14-Alkoxymethyl (demethoxy/CH)₂OR) (us patent 4,331,598);

(3) c-14-hydroxymethyl or acyloxymethyl (CH)₂OH or CH₂OAc) (U.S. patent 4,450,254) (prepared by Nocardia (Nocardia);

(4) c-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared from maytansinol by transformation with Streptomyces);

(5) c-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from the peach tree (Trewianudiflora));

(6) C-18-N-demethylation (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared from maytansinol by demethylation by Streptomyces; and

(7)4, 5-deoxy (U.S. Pat. No. 4,371,533) (prepared by titanium trichloride/LAH reduction of maytansinol).

In a preferred embodiment, the cytotoxic conjugates of the invention utilize thiol-containing maytansinoids (DM1), formally referred to as N, as the cytotoxic agent²' -Deacetyl N²' - (3-mercapto-1-oxopropyl) -maytansine. DM1 is represented by the following structural formula (I):

in another preferred embodiment, the cytotoxic agents of the invention utilize thiol-containing maytansinoids N²' -Deacetyl-N²' - (4-methyl-4-mercapto-1-oxopentyl) -maytansine as cytotoxic agent. DM4 is represented by the following structural formula (II):

in further embodiments of the invention, other maytansinoids may be used, including thiol and disulfide-containing maytansinoids having mono-or dialkyl substitution at the carbon atom bearing the sulfur atom. These include maytansinoids having an acylated amino acid side chain with a hindered sulfhydryl group on a C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl group, wherein the carbon atom of the acyl group bearing a thiol functionality has one or two substituents which are CH₃、C₂H₅Linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, cycloalkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl group, further wherein one of said substituents may be H, wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl function and the sulfur atom.

Such additional maytansinoids include compounds represented by formula (III):

wherein:

y' represents

(CR₇CR₈)_l(CR₉＝CR₁₀)_pC＝C_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C＝C)_sB_t(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl, and R₂May be H;

A. b, D is cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl or heteroaryl or heterocycloalkyl;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each independently is H, CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group;

l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not simultaneously 0; and

z is H, SR or-COR, where R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or simply a substituted aryl or heteroaryl or heterocycloalkyl group.

Preferred embodiments of formula (III) include compounds of formula (III),

wherein:

R₁is H, R₂Is methyl, Z is H;

R₁and R₂Is methyl, Z is H;

R₁is H, R₂Is methyl, Z is-SCH₃(ii) a Or

R₁And R₂Is methyl, Z is-SCH₃。

Such additional maytansinoids also include compounds represented by formula (IV-L), (IV-D) or (IV-D, L):

wherein:

y represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl, and R₂May be H;

R₃、R₄、R₅、R₇、R₇and R₈Each independently is H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0;

z is H, SR or-COR, wherein R is a linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, a cycloalkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aryl or heterocycloalkyl group; and

may represents maytansinoids with a side chain at the C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation.

Preferred embodiments of formulae (IV-L), (IV-D) and (IV-D, L) include compounds of formulae (IV-L), (IV-D) and (IV-D, L), wherein:

R₁is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0, and Z is H.

R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0, and Z is H.

R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0, and Z is-SCH₃。

R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0, and Z is-SCH₃。

Preferred cytotoxic agents are represented by formula (IV-L).

Such additional maytansinoids also include compounds represented by formula (V):

wherein:

y represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocycloaryl or heterocycloalkyl, and R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each independently is H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0; and

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group.

Preferred embodiments of formula (V) include compounds of formula (V), wherein:

R₁is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0, and Z is H.

R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0, and Z is H.

R₁Is H, R₂Is methyl，R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0, and Z is-SCH₃。

R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0, and Z is-SCH₃。

Such additional maytansinoids further include compounds represented by formula (VI-L), (VI-D) or (VI-D, L):

wherein:

Y₂represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocycloaryl or heterocycloalkyl, and R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each independently is H, CH₃、C₂H₅Linear cycloalkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0; and

Z₂is SR or COR, wherein R is a linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group; and

may is a maytansinoid.

Such additional maytansinoids also include compounds represented by formula (VII):

wherein:

Y₂' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C＝C)_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C＝C)_sB_t(CR₃CR₄)_nCR₁R₂ SZ₂，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cycloalkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocycloaryl or heterocycloalkyl, and R₂May be H;

A. b and D are each independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl, or a heterocyclic aromatic or heterocycloalkyl group;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each one of which isIndependently is H, CH₃、C₂H₅Linear alkyl or alkenyl groups having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl groups having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m, n, 0, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not simultaneously zero; and

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group.

Preferred embodiments of formula (VII) include compounds of formula (VII) wherein: r₁Is H and R₂Is methyl.

The above maytansinoids may be conjugated to anti-CA 6 antibody DS6 or a homologue or fragment thereof, wherein the antibody and maytansinoid are linked using a thiol or disulfide functional group present on the acyl group of the acylated amino acid side chain at the C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 dehydroxy of the maytansinoid, wherein the thiol or disulfide functional group present on the acyl group of the acylated amino acid side chain is on a carbon atom with one or two substituents being CH₃、C₂H₅Linear alkyl or alkenyl groups having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl groups having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups, and in addition, one of the substituents may be H, wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl function and the sulfur atom.

Preferred conjugates of the invention are conjugates comprising anti-CA 6 antibody DS6 or a homologue or fragment thereof linked to a maytansinoid of formula (VIII):

wherein:

Y₁' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C＝C)_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C＝C)_sB_t(CR₃CR₄)_nCR₁R₂ S-，

Wherein:

A. b and D are each independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl or heteroaryl group or a heterocycloalkyl group;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl group; and

l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not simultaneously zero.

Preferably, R₁Is H and R₂Is methyl or R₁And R₂Is methyl.

More preferred conjugates of the invention are conjugates comprising anti-CA 6 antibody DS6 or a homologue or fragment thereof, linked to a maytansinoid of formula (IX-L), (IX-D) or (IX-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic alkyl group, and further, R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl group;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0; and

may represents maytansinol, which has a side chain located at the C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation position.

Preferred embodiments of formulae (IX-L), (IX-D) and (IX-D, L) include compounds of formulae (IX-L), (IX-D) and (IX-D, L) wherein:

R₁is H and R₂Is methyl or R₁And R₂Is a methyl group, and the compound is,

R₁is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0,

R₁and R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are 1, and n is 0.

Preferably, the cytotoxic agent is represented by formula (IX-L).

Further preferred conjugates of the invention are conjugates comprising anti-CA 6 antibody DS6 or a homologue or fragment thereof, linked to a maytansinoid of formula (X):

wherein the substituents are as defined above for formula (IX).

Particularly preferred are any of the above compounds, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, and n is 0.

Further particularly preferred are any of the above compounds, wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, and n is 0.

Still further, the L-aminoacyl stereoisomer is preferred.

Various maytansinoids described in pending U.S. patent application Ser. No. 10/849,136, filed 5/20, 2004, may also be used in the cytotoxic conjugates of the present invention. The entire disclosure of U.S. patent application Ser. No. 10/849,136 is incorporated herein by reference.

Disulfide (disulphide) -containing linking groups

To link the maytansinoid to a cell-binding agent such as the DS6 antibody, the maytansinoid comprises a linking moiety. The linking moiety comprises a chemical bond that allows the release of the fully active maytansinoid at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds. Disulfide bonds are preferred.

The linking moiety also includes a reactive chemical group. In a preferred embodiment, the active chemical group may be covalently linked to the maytansinoid by a disulfide bond linking moiety.

Particularly preferred reactive chemical groups are N-succinimidyl esters (N-succinimidyl esters) and N-sulfosuccinimidyl esters (N-sulfosuccinimidyl esters).

Particularly preferred maytansinoids comprising a linking moiety comprising a reactive chemical group comprising an N-succinimidyl ester or an N-sulfosuccinimidyl ester are C-3 esters of maytansinol and analogues thereof, wherein the linking moiety comprises a disulfide bond.

Many positions on maytansinoids can serve as positions for chemical linking moieties. For example, the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group are all considered useful. However, the C-3 position is preferred, and the C-3 position of maytansinol is particularly preferred.

Although the synthesis of an ester of maytansinol with a linking moiety has been described in terms of a disulfide-containing linking moiety, one of ordinary skill in the art will appreciate that linking moieties with other chemical bonds (as described above) may also be used in the present invention, as may other maytansinoids. Specific examples of other chemical bonds include acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds. The disclosure of U.S. Pat. No. 5,208,020, incorporated herein, teaches the preparation of maytansinoids having such a linkage.

The synthesis of maytansinoids and maytansinoid derivatives having a disulfide moiety containing a reactive group is described in U.S. Pat. Nos. 6,441,163 and 6,333,410 and U.S. application Ser. No. 10/161,651, which are incorporated herein by reference.

Reactive group-containing maytansinoids, such as DM1, are reacted with antibodies, such as DS6 antibodies, to produce cytotoxic conjugates. These conjugates can be purified by HPLC or gel filtration.

Several good protocols for producing such antibody-maytansinoid conjugates are provided in U.S. Pat. No. 6,333,410 and U.S. application Ser. Nos. 09/867,598, 10/161,651, and 10/024,290, the entire contents of which are incorporated herein.

In general, a molar excess of maytansinoids having a disulfide moiety containing a reactive group can be incubated with a solution of the antibody in an aqueous buffer. The reaction compound is terminated by the addition of an excess of an amine (e.g., ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate was then purified by gel filtration.

The amount of maytansinoid molecules bound per antibody molecule can be determined by spectrophotometric measurement of the ratio of absorbance at 252nm and 280 nm. Preferably an average of 1-10 maytansinoid molecules per antibody molecule.

Conjugates of the antibodies with maytansinoids can be evaluated for their ability to inhibit the proliferation of various undesirable cell lines in vitro. For example, cell lines such as human epidermal cancer cell line a-431, human small cell lung cancer cell line (human breast cancer cell line) SW2, human breast tumor cell line (human breast tumor line) SKBR3, and burkitt lymphoma cell line Namalwa can be readily used to evaluate the cytotoxicity of these compounds. The cells to be evaluated can be exposed to the compound for 24 hours and the proportion of survival of the cells measured in a direct assay by known methods. From the measurement results, IC can then be calculated₅₀The value is obtained.

PEG-containing linking groups

PEG linkers can also be used to link the maytansinoids to the cell-binding agent, as described in U.S. application Ser. No. 10/024,290. These PEG linking groups, which are soluble in both water and non-aqueous solvents, can be used to link one or more cytotoxic agents to a cell binding agent. Exemplary PEG linking groups include heterobifunctional PEG linkers that attach cytotoxic agents and cell binding agents at both ends of the linker through a functional thiol or disulfide group at one end and an active ester at the other end.

As a general example of the synthesis of cytotoxic conjugates using PEG linking groups, reference may also be made to U.S. application Ser. No. 10/024,290 for specific details thereof. Synthesis begins by reacting one or more cytotoxic agents having active PEG moieties with a cell-binding agent, resulting in the replacement of the terminal active ester of each active PEG moiety by an amino acid residue of the cell-binding agent, thereby producing a cytotoxic conjugate comprising one or more cytotoxic agents covalently linked to the cell-binding agent via a PEG linking group.

Taxanes (Taxanes)

The cytotoxic agent used in the cytotoxic conjugate of the present invention may also be a taxane or a derivative thereof.

Taxanes are a class of compounds including paclitaxel (Taxol), which is a cytotoxic natural product, and docetaxel (Taxotere), which is a semi-synthetic derivative, both of which are widely used in the treatment of cancer. Taxanes are mitotic spindle poisons that inhibit the depolymerization of tubulin, leading to cell death. Although docetaxel and paclitaxel are useful agents in the treatment of cancer, their antitumor activity is limited due to their non-specific toxicity to normal cells. Furthermore, compounds like paclitaxel and docetaxel by themselves are not sufficiently effective for use in conjugates with cell binding agents.

Preferred taxanes that can be used to prepare the cytotoxic conjugates are taxanes of formula (XI):

methods for the synthesis of taxanes that may be used in the cytotoxic conjugates of the invention, as well as methods for conjugation of taxanes with cytotoxic agents such as antibodies, are described in detail in U.S. Pat. Nos. 5,416,064, 5,475,092, 6,340,701, 6,372,738, and 6,436,931, and in U.S. application Ser. Nos. 10/024,290, 10/144,042, 10/207,814, 10/210,112, and 10/369,563.

CC-1065 analogs

The cytotoxic agent employed in the cytotoxic conjugate of the present invention may also be CC-1065 or a derivative thereof.

CC-1065 is a potent antitumor antibiotic isolated from a culture of Streptomyces zelensis. CC-1065 has about 1000 times higher potency in vitro than commonly used anticancer drugs such as doxorubicin (doxorubicin), methotrexate (methotrexate) and vincristine (vincristine) (B.K. Bhuyan et al, Cancer Res., 42, 3532-3537 (1982)). CC-1065 and its analogs are disclosed in U.S. Pat. Nos. 6,372,738, 6,340,701, 5,846,545, and 5,585,499.

The cytotoxic potency (cytotoxic potency) of CC-1065 is related to its alkylating activity and its DNA binding or DNA intercalating activity. These two activities are located in different parts of the molecule. Thus, the alkylation activity is contained in the Cyclopropylpyrroloindole (CPI) subunit and the DNA binding activity is located in both pyrroloindole (pyrroloidolondole) subunits.

Although CC-1065 has certain attractive features as a cytotoxic agent, it has limitations in its therapeutic use. Administration of CC-1065 to mice causes delayed hepatotoxicity, which results in death at day 50 after a single intravenous dose of 12.5. mu.g/kg { V.L.Reynolds et al, J.antibiotics, XXIX, 319-334(1986) }. This has motivated efforts to develop analogs that do not cause delayed poisoning, and the synthesis of simpler analogs modeled as CC-1065 has been described { m.a. warpehoski et al, j.med.chem., 31, 590-603(1988) }.

In another series of analogues, the CPI moiety is replaced by a Cyclopropapabenzindole (CBI) moiety { D.L.Boger et al, J.Org.Chem., 55, 5823-5833, (1990), D.L.Boger et al, BioOrg.Med.Chem.Lett., 1, 115-120(1991) }. These compounds maintain high in vitro potency of the parent drug without inducing delayed toxicity to mice. Like CC-1065, these compounds are alkylating agents that bind covalently to the minor groove of DNA to cause cell death. However, clinical evaluation of the most promising analogues, Adozelesin (Adozelesin) and kazelesin (Carzelesin), has yielded disappointing results { b.f. foster et al, Investigational New Drugs, 13, 321-326 (1996); wolff et al, Clin. cancer Res., 2, 1717-1723(1996) }. These drugs show poor therapeutic effect because of high systemic toxicity.

The therapeutic efficacy of CC-1065 analogs can be greatly improved by targeting delivery to the tumor site to alter their distribution in vivo, thus having lower toxicity to non-target tissues and therefore lower systemic toxicity. To achieve this goal, conjugates of analogs and derivatives of CC-1065 with cell-binding agents that specifically target tumor cells have been described { U.S. patent: 5,475,092; 5,585,499, respectively; 5,846,545}. These conjugates typically show high target-specific cytotoxicity in vitro and extraordinary antitumor activity in a human tumor xenograft model in mice { r.v.j.chari et al, Cancer res, 55, 4079-4084(1995) }.

Methods for the synthesis of CC-1065 analogs useful in the cytotoxic conjugates of the invention, as well as methods for conjugation of the analogs to cell binding agents such as antibodies, are described in detail in U.S. Pat. Nos. 5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618, and in U.S. applications Ser. Nos. 10/116,053 and 10/265,452.

Other drugs

Drugs such as methotrexate (methotrexate), daunorubicin (daunorubicin), doxorubicin (doxorubicin), vincristine (vincristine), vinblastine (vinblastine), melphalan (melphalan), mitomycin C (mitomycin C), chlorambucil (chlorembucil), calicheamicin (calicheamicin), tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs, dolastatin and dolastatin analogs are also suitable for preparing the conjugates of the invention. The drug molecules may be linked to the antibody molecules by intermediate carrier molecules such as serum albumin. For example, Doxarubicin and Danorubicin compounds may also be useful cytotoxic agents, as described in U.S. Ser. No. 09/740991.

Therapeutic compositions

The present invention also provides a therapeutic composition comprising:

(a) an effective amount of one or more cytotoxic conjugates; and

(b) a pharmaceutically acceptable carrier.

Likewise, the invention provides a method for inhibiting the growth of a particular cell population, the method comprising contacting a target cell or tissue comprising the target cell with an effective amount of a cytotoxic conjugate or a therapeutic agent comprising a cytotoxic conjugate, either administered alone or in combination with other cytotoxic or therapeutic agents.

The invention also includes methods of treating a subject having cancer using the therapeutic compositions of the invention.

Cytotoxic conjugates can be evaluated for potency and specificity in vitro by methods previously described (see, e.g., R.V.J.Chari et al, Cancer Res.55: 4079-4084 (1995)). Antitumor activity can be assessed in a mouse human tumor xenograft model by methods also previously described (see, e.g., Liu et al Proc. Natl. Acad. Sci.93: 8618-8623 (1996)).

Suitable pharmaceutically acceptable carriers are well known and can be determined by one of ordinary skill in the art to meet clinical criteria. As used herein, carriers include diluents and excipients.

Examples of suitable carriers, diluents and/or excipients include: (1) dulbecco phosphate buffered saline, pH-7.4, with or without about 1mg/ml to 25mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) glucose; antioxidants such as tryptamine and stabilizers such as Tween 20 may also be included.

Methods of inhibiting the growth of a selected cell population can be practiced in vitro (in vitro), in vivo (in vivo), or ex vivo (ex vivo). As used herein, inhibiting growth means slowing the growth of cells, reducing cell viability, causing cell death, lysing cells, and inducing cell death, whether in a short period of time or a long period of time.

Examples of in vitro uses include the treatment of autologous bone marrow prior to transplantation into the same patient in order to kill diseased or malignant cells; treatment prior to bone marrow transplantation for the purpose of killing activated T cells (component T cells) and preventing Graft Versus Host Disease (GVHD); treatment of the cell culture with the aim of killing all cells except the desired variant which does not express the target antigen; or killing variants that express undesired antigens.

One of ordinary skill in the art can readily determine the circumstances of non-clinical in vitro applications.

Examples of ex vivo clinical applications are the removal of tumor cells or lymphocytes from bone marrow prior to autologous transplantation, or the removal of T cells and other lymphocytes from autologous or allogeneic bone marrow prior to transplantation, in the treatment of cancer or autoimmune diseases, with the aim of preventing Graft Versus Host Disease (GVHD). Treatment may be performed as follows. Bone marrow is harvested from a patient or other individual and then incubated in a medium containing serum and added with the cytotoxic agent of the present invention. The concentration is between about 10. mu.M to 1pM, and the incubation is at about 37 ℃ for about 30 minutes to about 48 hours. The concentration and incubation time of the specific conditions, i.e. dosage, can be easily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned to the patient by intravenous infusion according to known methods. In some cases, during the period between harvesting of the bone marrow and reinfusion of the treated cells, the patient receives other treatments such as ablative chemotherapy or whole body radiation, at which time the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

For clinical use in vivo, the cytotoxic conjugates of the invention will be supplied in solution, which will be tested for sterility and endotoxin levels. Examples of suitable administration regimens for the cytotoxic conjugate are as follows. The conjugate was administered weekly in an intravenous bolus for 4 weeks. The bolus dose is provided in 50 to 100ml of physiological saline to which 5 to 10ml of human serum albumin may be added. The dose is 10. mu.g to 100mg per administration, intravenously (100ng to 1mg/kg per day). More preferably, the dose is between 50 μ g and 30 mg. Most preferably, the dose is between 1mg and 20 mg. After four weeks of treatment, the patient may continue to receive treatment on a weekly basis. The skilled artisan can determine the particular clinical regimen regarding the route of administration, excipients, diluents, dosage, number of times, etc., as clinical circumstances warrant.

Depending on the in vivo or ex vivo method of killing the selected cell population, examples of medical conditions that may be treated include any type of malignant condition, including, for example, lung, breast, colon, prostate, kidney, pancreas, ovary, cervix and lymphatic organ cancers, osteosarcoma, synovial cancer, sarcomas or carcinomas in which CA6 is expressed and other cancers to be identified in which the CA6 carbohydrate epitope is significantly expressed; autoimmune diseases such as systemic lupus (systemic lupus), rheumatoid arthritis (rheumatoid arthritis) and multiple sclerosis; graft rejection reactions such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; graft versus host disease; viral infections, such as mV infection, HIV infection, AIDS, and the like; and parasitic infections such as giardiasis, amebiasis, schistosomiasis, and others as determined by one of ordinary skill in the art.

Reagent kit

The invention also includes kits, e.g., comprising the cytotoxic conjugates and instructions for using the cytotoxic conjugates to kill a particular cell type. The instructions may include directions for using the cytotoxic conjugate in vitro, in vivo, or ex vivo.

Typically, the kit has a separate part (component) containing the cytotoxic conjugate. The cytotoxic conjugate may be in a lyophilized form, a liquid form, or other form suitable for inclusion in a kit. The kit may also contain additional components necessary for carrying out the methods described on the instructions for use in the kit, such as sterile solutions for reconstituting the lyophilized powder, additional reagents for combining with the cytotoxic conjugate prior to administration to the patient, and means to facilitate administration of the conjugate to the patient.

Other embodiments

The invention further provides monoclonal antibodies, humanized antibodies and epitope-binding fragments thereof, which are further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion.

Diagnostic methods are also provided wherein the labeled antibody or epitope-binding fragment thereof is administered to a subject suspected of having cancer and the distribution of the label in the subject is measured or monitored.

Examples

The broad scope of the invention can be best understood by reference to the following examples, which are not intended to limit the invention to the specific embodiments.

Example 1: identification of antigen positive and negative cells by flow cytometry binding assays

Flow cytometry was used to localize the DS6 epitope CA6 to the cell surface. Human cell lines were obtained from the American Type Culture Collection (ATCC) except OVCAR5(Kearse et al, int. J. cancer 88 (6): 866-872(2000)), OVCAR8, and IGROV1 cells (M.Seiden, Massachusetts general Hospital). All cells were cultured in RPMI 1640 supplemented with 4mM L-glutamine, 50U/ml penicillin, 50. mu.g/ml streptomycin (Cambrex Bio Science, Rockland, ME) and 10% v/v fetal bovine serum (Atlas Biologicals, Fort Collins, CO), which is referred to as medium hereinafter. Cells were maintained at 37C, 5% CO₂A humid incubator.

Cells (1-2X 10) were incubated with DS6 antibody at serial dilution concentrations prepared in FACS buffer (2% goat serum, RPMI) in 96-well plates⁵Individual cells/well) were incubated on ice for 3-4 h. Cells were spun down at 1500rpm in a bench top centrifuge for 5 minutes at 4 ℃. After removal of the medium, the wells were refilled with 150. mu.l of FACS buffer. The washing step is then repeated. FITC-labeled goat anti-mouse IgG (Jackson hnmunoresearch) was diluted 1: 100 in FACS buffer and incubated with cells on ice for 1 h. The cell culture plates were covered with foil to prevent photobleaching of the signal. After two washes, cells were fixed with 1% formaldehyde and analyzed on a flow cytometer.

Notably, the CA6 epitope is found in ovary, breast, uterusIn cell lines of cervical and pancreatic origin (table 3), as predicted by tumor immunohistochemistry (immunohistochemistry). However, some cell lines of other tumor types showed limited CA6 expression. DS6 antibody binds with an apparent K of 135.6pM_D(in PC-3 cells, Table 3). In antigen positive cell lines, the maximum mean fluorescence (table 3) of the binding curve (fig. 1) represents the relative antigen density.

TABLE 3

^*Mean maximum relative mean fluorescence

Example 2: characterization of the DS6 epitope

The properties of the DS6 antigen, CA6, were analyzed by immunoblotting of a dot blot of CA6 positive cell lysate (Caov-3) digested with proteolytic (pronase and proteinase K) and/or sugar cleavage (neuraminidase and periodic acid). For positive controls, other antibodies recognizing various epitope types were tested on antigen positive cell lines (Caov-3 and CM 1; Colo205 and C242; SKMEL28 and R24). CM1 is an antibody that recognizes a protein epitope of an indeterminate number of tandem repeat domains (VNTRs) of Muc-1, thus providing a control for the protein epitope. C242 binds to a novel colorectal cancer specific sialic acid dependent carbohydrate epitope (CanAg) on Muc-1, which provides a control for carbohydrate epitopes (glycotopes) on proteins. R24 binds to GD3 ganglioside specific for melanoma, thus providing a control for carbohydrate epitopes on the non-protein backbone.

Caov-3, Colo205 and SKMEL28 cells were plated in 15cm tissue culture plates. The day before lysis, the medium was refreshed (30 mL/plate). Modified RIPA buffer (50mM Tris-HCl ph7.6, 150mM NaCl, 5mM EDTA, 1% NP40, 0.25% sodium deoxycholate), protease inhibitors (PMSF, pepstatin a, leupeptin and aprotinin) and PBS were pre-cooled on ice. After aspirating the medium from the plate, the cells were washed twice with 10ml of cold PBS. All subsequent steps were performed on ice and/or in a 4 ℃ cold room. After the final wash of PBS was aspirated, the cells were lysed with 1-2mL lysis buffer (RIPA buffer with fresh protease inhibitor added, final concentration of 1mM PMSF, 1. mu.M pepstatin A, 10. mu.g/mL leupeptin and 2. mu.g/mL aprotinin). The lysate was scraped off the plate using a cell harvester (cell lifter) and the lysate was crushed by pipetting the suspension up and down (5-10 times) using an 18G needle. The lysate was vortexed for 10 minutes and then centrifuged in a microcentrifuge for 10 minutes at maximum speed (13K rpm). The pellet was removed and the supernatant was assayed using the Bradford protein concentration assay kit (Biorad).

The lysate (2. mu.l) was directly transferred onto a dry 0.2 μm nitrocellulose membrane. The spots were allowed to air dry for approximately 30 minutes. The film is cut into pieces, each piece containing one dot. At 37 ℃ in the presence of pronase (1mg/ml enzyme, 50mM Tris pH7.5, 5mM CaCl)₂) Proteinase K (1mg/ml enzyme, 50mM Tris pH7.5, 5mM CaCl)₂) Neuraminidase (20mU/ml enzyme, 50mM sodium acetate pH5, 5mM CaCl)₂100. mu.g/ml BSA) or periodic acid (20mM, 0.5M sodium acetate pH5) for 1 h. Reagents were purchased from Roche (enzyme) and VWR (periodic acid). Membranes were washed in T-TBS wash buffer (0.1% Tween 20, 1XTBS) (5 min), blocked with blocking buffer (3% BSA, T-TBS) at room temperature for 2h, and incubated overnight at 4 ℃ in blocking buffer with 2. mu.g/ml primary antibody (i.e., DS6, CM1, C242, R24). The membrane was washed three times with T-TBS for 5 minutes and then incubated with HRP conjugated goat anti-mouse (or human) IgG secondary antibody (Jackson Immunoresearch; 1: 2000 dilution in blocking solution) for 1h at room temperature. Washing the immunoblotWashed three times and developed using ECL system (Amersham).

Immunoblotting (fig. 2) of the digested control lysates showed that the CM1 signal was disrupted by proteolytic processing, whereas the signal of the sugar cleaved digest was unaffected, which was expected for antibodies recognizing protein epitopes. The C242 signal is disrupted by proteolytic or carbohydrate cleavage treatment, which is expected for antibodies that recognize carbohydrate epitopes found on proteins. The R24 signal was unaffected by proteolytic processing, and was completely disrupted by neuraminidase or periodic acid treatment, as would be expected for antibodies recognizing gangliosides. DS6 immunoblots dot blotted with digested Caov-3 lysate showed a loss of signal after treatment with proteolytic and carbohydrate cleavage compounds. Thus, like C242, DS6 binds to a carbohydrate epitope on the protein core. In addition, the signal in the DS6 immunoblot was sensitive to neuraminidase treatment. Thus, CA6 is a sialic acid-dependent carbohydrate epitope like CanAg.

To confirm the carbohydrate nature of CA6, the Caov-3 cleavage product was spotted onto a PVDF membrane and treated with a chemical deglycosylating reagent, trifluoromethanesulfonic acid (TFMSA), under nitrogen at room temperature for 5 minutes. Dot blots were washed with T-TBS and immunoblotted with CM1 or DS6 (FIG. 3). The DS6 signal was disrupted after treatment with acid, which provides further evidence that CA6 is a carbohydrate epitope. CM1 signal increased after TFSMA treatment, indicating that the acid treatment did not affect the protein on the membrane, and that the sugar cleavage treatment exposed the protein epitope recognized by CM 1.

To further illustrate the structure of the CA 6-containing sugars, dot blots were digested with N-glycanase, O-glycanase, and/or sialidase (fig. 4). Caov-3 cell lysates (100. mu.g, 30. mu.l) were incubated with 2.5. mu.l of denaturing buffer containing SDS and beta-mercaptoethanol (Glyko) for 5 min at 100 ℃. The denatured cleavage products were then digested with 1. mu.l N-glycanase, O-glycanase and/or sialidase A (Glyko) for 1h at 37 ℃. The digested lysate was then spotted (2. mu.l) onto nitrocellulose and immunoblotted as above.

N-glycanase had no significant effect on DS6 immunoblot signal. However, the samples digested with sialidase did not produce a signal. Since the O-glycanase cannot digest sialylated O-linked saccharides that have not been pre-treated with sialidase, the DS6 signal of samples processed with O-glycanase alone will not be affected. In contrast, the activity of N-glycanase was independent of any glycosidase pretreatment. The fact that the DS6 signal was not affected by the N-glycanase treatment indicates that the CA6 epitope is most likely located on sialylated O-linked sugar chains.

Example 3: elucidation of antigens on which the CA6 epitope is found

To identify the antigen on which the CA6 sialyl epitope was found, the DS6 immunoprecipitation reaction was analyzed by SDS-PAGE and Western blotting. Cell lysate supernatant (1 mL/sample; 3-5mg Protein) was pre-cleared with Protein G beads (30. mu.l) equilibrated with 1mL RIPA buffer for 1-2h at 4 ℃ with rotation. All subsequent steps were performed on ice and/or in a 4 ℃ cold room. The beads were spun down in a microcentrifuge for a short period of time to pellet precleans (2-3 s). The pretreated supernatant was transferred to a clean tube and incubated overnight with 2 μ g of ds6 under rotation. Freshly prepared equilibrated protein G beads (30. mu.l) were added to the lysate and incubated for 1h under rotation. The sedimented bead-lysate suspension is spun for a short time in a microcentrifuge, and a sample of the lysate after the immunoprecipitation reaction can optionally be removed. The beads were washed 5-10 times with 1mL of RIPA buffer.

Then 30. mu.l neuraminidase (20mU neuraminidase (Roche), 50mM sodium acetate pH5, 5mM CaCl) at 37 ℃₂100. mu.g/ml BSA) or 30. mu.l periodic acid (20mM periodic acid (VWR), 0.5M sodium acetate pH5) the immunoprecipitated DS6 samples were digested for 1 h. This was then suspended in 30. mu.l of 2 Xsample loading buffer (containing. beta. -mercaptoethanol). The beads were boiled for 5 minutes and the loading buffer supernatant was loaded onto a 4-12% or 4-20% Tris-glycine gel (Invitrogen). At 125V, theThe gels were separated in Laemmli electrophoresis buffer (Laemmli electrophoresis running buffer) for 1.5 h. The gel samples were transferred onto 0.2 μm nitrocellulose membranes (Invitrogen) overnight at 20mA using a MiniTrans-blot transfer apparatus (Biorad). This membrane was immunoblotted with DS6 as described in example 2 above.

Alternatively, the immunoprecipitated beads were first denatured and then enzymatically digested with N-glycanase, O-glycanase and/or sialidase A (Glyko). The beads were resuspended in 27. mu.l incubation buffer and 2. mu.l denaturation buffer (Glyko) and incubated at 100 ℃ for 5 minutes. After cooling to room temperature, detergent solution (2. mu.l) was added and the samples were incubated with N-glycanase, O-glycanase and/or sialidase A for 4 hours at 37 ℃. After adding 5 Xsample loading buffer (7. mu.l), the sample was boiled for 5 minutes. Samples were subjected to SDS-PAGE and immunoblotting as described above.

DS6 immunoprecipitated a band of > 250kDa, a band of this protein seen in antigen-positive cell lysates (FIGS. 5A, B and C). In some cell lines (i.e., T-47D), two bands (doubls) were observed. In the Caov-3 immunoprecipitation reaction treated with neuraminidase or periodic acid (FIGS. 5A and B), the band at > 250kDa was disrupted, indicating that the CA6 epitope is located on the band at > 250 kDa. Bands > 250kDa were also demonstrated to be insensitive to immunoprecipitated N-glycanase treatment, which is consistent with CA6 being on the O-glycoconjugate (FIG. 5F). Furthermore, the support for the 250kDa band as CA6 antigen resides in the fact that DS6 immunoprecipitation did not show such a band in DS6 antigen-negative cells (FIGS. 5D and E).

There is several lines of evidence that the CA6 antigen is Muc 1. The CA6 antigen is likely to be mucin due to its high molecular weight and sensitivity to O-linked carbohydrate specific glycohydrolases. Mucin overexpression is well characterized in tumors, particularly in breast and ovarian tumors, consistent with the major tumor reactivity of DS 6. Furthermore, like CanAg (which is the sialyl epitope on Muc 1), CA6 is insensitive to perchloric acid precipitation, suggesting that the CA6 antigen is most likely (heavily) O-glycosylated. It was observed that in some DS6 expressing cell lines, DS6 immunoprecipitated two bands of > 250kDa, suggesting that CA6 was Muc 1. Human Muc1 is characterized by the presence of two different Muc1 alleles with different numbers of tandem repeats, which results in the expression of two Muc1 proteins of two different molecular weights.

To test whether CA6 was found on Muc1, DS6 immunoprecipitates from Caov-3 lysates were subjected to SDS-PAGE and immunoblotted with DS6 or the Muc1 VNTR antibody CM 1. As observed in fig. 6A, CM1 reacted strongly with the > 250kDa band immunoprecipitated from DS 6. In FIG. 6B, DS6 and CM1 immunoprecipitates from HeLa cell lysates showed the same two bands > 250kDa when immunoblotted with DS6 or CM 1. These results indicate that the CA6 epitope is indeed located on the Muc-1 protein. The double band of DS6 observed in HeLa (and T-47D) cells can be explained by the fact that expression of Muc-1 is directed by different alleles with different numbers of tandem repeats.

Although CM1 and DS6 bind the same Muc-1 protein, they are distinct epitopes. Dot blot of Caov-3 lysate completely disrupted the DS6 signal by chemical deglycosylation of trifluoromethanesulfonic acid (TFMSA) (FIG. 3). However, the same process enhances the CM1 signal. Deglycosylation may be the exposure of the hidden CM1 antibody epitope. Furthermore, comparison of the flow cytometry binding results of DS6 and CM1 (fig. 4) indicated that the CA6 epitope was not present on every cell expressing Muc 1. Interestingly, the CA6 epitope was not expressed on Colo205 (table 3), whereas the Colo205 cell line was known to express high levels of the Muc1CanAg sialyl epitope.

TABLE 4

^*Maximum mean relative fluorescence

Example 4: quantitative analysis of the shed (bred) CA6 epitope

Since the CA6 epitope is located on Muc1, it is known that in many cancer patients, the Muc1 molecule will extravasate into the blood vessels, and quantitative methods were taken with the aim of determining whether such levels would inhibit DS6 antibody therapy. Binding of circulating antibodies to antigens is thought to result in rapid clearance of immune complexes from the blood. If a significant portion of the administered antibody dose is rapidly removed from circulation, the amount reaching the tumor is likely to decrease, resulting in a decrease in the anti-tumor activity of the antibody therapeutic. When antibodies are conjugated to highly potent cytotoxic compounds, rapid clearance of the conjugate can potentially increase non-specific toxicity. Thus, for antibody-small drug conjugates, such as DS6-DM1, it is expected that high levels of shed antigen will reduce the anti-tumor effect and increase dose-limiting toxicity.

Recent clinical trials of antibody therapeutics have gained relevant information regarding the impact of shed antigen concentration on pharmacokinetics. For example, in clinical trials of trastuzumab (Herceptin), an antibody used to treat Her 2/neu-expressing metastatic breast cancer, the pharmacokinetics of trastuzumab clearance appears to be unchanged when shed Her2/neu levels are less than 500ng/mL (Pegram et al, j.clin.oncol.16 (8): 2659-71(1998) assuming a shed Her2/neu molecular weight of 110,000 daltons, and shed Her2/neu molar concentrations below 4.5nM appear to have little effect on pharmacokinetics.

In another example, clinical trials using cantuzumab mertansine (huC242-DM1) showed that the level of shed CanAg (the C242 epitope) prior to treatment was not associated with the pharmacokinetics of antibody clearance (Tolcher et al, J.Clin. Oncol.21 (2): 211-22 (2003). CanAg epitope, similar to the CA6 epitope recognized by DS6, is a unique tumor-specific O-linked sialoglycosyl epitope on Muc 1. however, the heterogeneous nature of CanAg epitopes makes it difficult to quantify it in molar terms. in the general population, the Muc1 allele varies in length depending on the number of tandem repeats in an indeterminate number tandem repeat (VNTR) domain. The proportion of CanAg epitopes per Muc1 molecule will be different in a cohort of patients. For this reason, serum samples were measured for shed CanAg by sandwich ELISA, where shed Muc1 bearing a CanAg epitope was captured by C242 and detected by a biotinylated C242/streptavidin HRP system. Shed CanAg was quantified in normalized units (U) proportional to the number of epitopes per ml of serum, rather than expressed by the molarity of Muc 1. Similarly, there was a similar situation for quantification of the shed CA6 epitope. In contrast, for trastuzumab, there is only one epitope per wandering her2/neu molecule, which greatly simplifies quantitative determination of wandering antigen.

To link CA6 shedding epitope levels with those found in clinical trials of trastuzumab and cantuzumab mertansine, a method was developed for obtaining molar concentrations of complex shedding epitopes on Muc1, such as sialylated carbohydrate epitopes. First, a simple sandwich ELISA assay for DS6 was established. A depiction of this analysis is shown in fig. 7A. DS6 was used to capture Muc1 with the CA6 epitope. Because each Muc1 molecule has multiple CA6 epitopes, biotinylated DS6 was also used as a tracer antibody. Biotinylated DS6 bound to captured CA6 was detected with streptavidin-HRP using ABTS as substrate. The captured CA6 epitope was derived from ovarian cancer patient serum or from a standard commercially available Muc1 test kit (CA15-3) that was used to monitor shed Muc1 in breast cancer patients. DS6 units/ml was arbitrarily set, which is equivalent to CA15-3 standard units/ml.

The results of the DS6 sandwich ELISA are shown in FIG. 7B, where the CA15-3 standard was used. The resulting curves were very similar to those obtained with the CA15-3 standard in the CA15-3 analysis. To convert DS6 units/ml to CA6 molarity, a biotinylated DS6 standard curve converting the signal to picogram DS6 was required. Assuming one-to-one stoichiometry between the CA6 epitope and the biotinylated DS6 antibody, and a molecular weight of 160,000 daltons for biotinylated DS6, the moles of CA6 captured per volume of sample added can be calculated.

Two alternative methods for generating a standard curve representing biotinylated DS6 are depicted in fig. 8A and B. In fig. 8A, goat anti-mouse IgG polyclonal antibody was used to capture biotinylated DS6, which was then detected in the same manner as used in the sandwich ELISA assay shown in fig. 7. In the method shown in fig. 8B, biotinylated DS6 was placed directly onto the ELISA plate and detected as shown in fig. 8A. As seen in fig. 8C, the standard curve of biotinylated DS6 generated by each method was very consistent.

The analysis of various shedding antigens from serum samples from ovarian cancer patients is shown in Table 5. The CA125 ELISA is typically used to monitor treatment of ovarian cancer patients by measuring shed CA125 units/ml. The CA125 status of the serum sample is provided. The CA15-3 ELISA is generally used to monitor treatment of breast cancer patients by measuring the units/ml of shed Muc1 using capture and detection antibodies that recognize epitopes different from those recognized by DS 6. In Table 5, CA15-3 was measured in serum samples from ovarian cancer patients.

TABLE 5

¹Measured by commercial ELISA kit

²Determined by the commercial CA15-3 standard (1 CA 15-3U ═ 1 DS 6U)

³Goat anti-mouse IgG&Biotin-DS 6 standard curve

⁴Biotin-DS 6 standard curve

For the values of CA15-3 reported in Table 5, the commercially available CA15-3 Enzyme immunoassay kit (Enzyme Immuno Assay kit) from CanAg Diagnostics was used. For DS6 units/ml, a CA15-3 standard (CA15-3 enzyme immunoassay kit from CanAg Diagnostics) was used to generate a standard curve in a DS6 sandwich ELISA. DS6 units/ml was arbitrarily set equal to CA15-3 units/ml. In the last two columns, picomolar (pM) flowoff CA6 was calculated using the biotinylated DS6 standard curve shown in fig. 8C.

With respect to quantitative analysis of CanAg levels, reported serum levels of CanAg were for patients who participated in the Cantuzumab mertansine clinical trial prior to treatment (Tolcher et al, J. Clin. Oncol.21 (2): 211-22 (2003); Using CanAg standards, Using an ELISA assay similar to that described for DS6, a CanAg standard curve was made C242 was used to capture the CanAg standard, biotinylated C242 tracer was used, followed by developing with streptavidin-HRP using ABTS as a substrate to complete the detection of captured CanAg, a biotinylated C242 standard curve was established, as was done for biotinylated DS6, to convert units/ml to molar concentrations of circulating CanAg epitopes. in Table 6, CanAg levels from patients in the Cantuzumab mertansine clinical trial were reported, along with the corresponding calculated molar concentrations of circulating CanAg.

TABLE 6

¹Pre-treatment levels of circulating CanAg as measured by sandwich ELISA

²Goat anti-mouse IgG&Biotin-C242 standard curve

³Biotin-C242 standard curve

Comparison of the pM levels of shed CA6 in ovarian cancer patients with calculated pM levels of shed CanAg in CanAg-positive cancer patients shows that shed CA6 levels are generally similar to shed CanAg levels. Furthermore, only 2 of the serum samples from 16 ovarian cancer patients were likely to have CA6 levels greater than 4.5nM, (serum samples 5 and 9, whose signals were outside the range of the standard curve), beyond which it was observed in clinical trials with Her2/neu positive breast cancer patients that altered herceptin pharmacokinetics. Of the 37 clinical trial patients, only 3 were found to have CanAg levels above 4.5 nM. In this clinical trial, shed CanAg levels were not associated with faster clearance of cantuzumab mertansine. However, patients with the highest CanAg level (31240U/ml) were only sampled 8 hours after transfusion. These results indicate that some epitopes of Muc1, such as CA6 and CanAg, although shed in cancer patients, shed levels that do not reach levels that inhibit treatment with antibody therapeutics.

Example 6: cloning of murine DS6 antibody variable regions

In clinical situations, murine monoclonal antibodies such as DS6 have limited utility because they are recognized as foreign by the human immune system. The patient will develop human anti-mouse antibodies (HAMA) quickly, which results in rapid clearance of the murine antibodies. Thus, the variable region of murine DS6(muDS6) was surface-reconstructed to generate humanized DS6(huDS6) antibody.

The murine DS6 antibody variable regions were cloned by RT-PCR. Total RNA was purified from confluent T175 flasks of DS6 hybridoma cells using Qiagen RNeasy mini-prep kit. The RNA concentration was determined by UV spectrophotometry using a Gibco Superscript II kit and random hexamer primers (random hexamer primers) to perform RT reactions with 4-5. mu.g total RNA.

PCR reactions were performed with degenerate primers based on Wang Z et al, J Immunol methods, jan 13; 233(1-2): 167-77 (2000). The RT reaction mixture was used directly for degenerate PCR reactions. The 3' light chain primer HindKL was used,

(TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC)(SEQ IDNO：25)

and a 3' heavy chain primer BamHI G1,

(GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC)(SEQ ID NO：26)，

for the 5' PCR primer, the light chain primer was SaclmK

(GGGAGCTCGAYATTGTGMTSACMCARWCTMCA)(SEQ ID NO：27)，

Heavy chain primers are

EcoR1MH1(CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC)(SEQ ID NO：28)

And

EcoR1MH2(CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG) (SEQ ID NO: 29) in equal mixtures (mixed bases: H ═ a + T + C, S ═ G + C, Y ═ C + T, K ═ G + T, M ═ a + C, R ═ a + G, W ═ a + T, V ═ a + C + G, N ═ a + T + G + C).

The PCR reaction was standard, except that the PCR reaction was supplemented with 10% DMSO (50. mu.l reaction mixture containing the final concentrations of 1 Xreaction buffer (ROCHE), 2mM each dNTP, 1mM each primer, 2. mu.l RT reaction product, 5. mu.l DMSO and 0.5. mu.l Taq (ROCHE)). The PCR reaction was carried out on an MJ research thermocycler (research thermocycler) using a procedure modified from Wang Z et al, (J immunological methods. Jan 13; 233 (1-2): 167-77 (2000)): 1)94 ℃ for 3 minutes; 2) 15 seconds at 94 ℃; 3) 1 minute at 45 ℃; 4)72 ℃ for 2 minutes; 5) loop back to step #229 times; 6) the final extension step was ended at 72 ℃ for 10 minutes. The PCR product was cloned into pBluescript II SK + (Stratagene) using restriction enzymes through restriction sites created by PCR primers. The Seqwright sequencing service sequenced heavy and light chain clones.

To confirm the 5' end cDNA sequence, additional PCR and cloning were performed. The DS6 light and heavy chain cDNA sequences determined by degenerate PCR cloning were placed into the Blast search site at NCBI and the murine antibody sequences with the submitted signal sequences were saved. PCR primers were designed from these signal peptides using conserved peptide stretches in the relevant DNA sequences. EcoRI restriction sites were added to the leader sequence primers (Table 7) and these were used in the RT-PCR reaction as described above.

TABLE 7

Several individual light and heavy chain clones were sequenced to identify and avoid possible polymerase-generated sequence errors. For both light and heavy chain RT-PCR clones, only a single sequence was obtained. These sequences were sufficient for designing primers that could amplify the murine DS6 light and heavy chain sequences that extended into the signal sequence. Subsequent clones from these subsequent PCR reactions confirmed the 5' terminal sequence of the variable region, which was altered by the original degenerate primers. The cumulative results from the various cDNA clones provided the final murine DS6 light and heavy chain sequences presented in fig. 9. Using Kabat and AbM definitions, three light and heavy chain CDRs were identified (fig. 9 and 10). A search of the NCBI IgBlast database showed that the heavy chain variable region of the muDS6 antibody is most likely derived from the murine IgVap4 germline gene and the heavy chain variable region is most likely derived from the murine IgVh J558.41 germline gene (fig. 11).

Example 7: determination of surface residues of the variable region of the DS6 antibody

Antibody surface reconstruction techniques described by Pedersen et al (1994) and Roguska et al (1996) begin with predicting surface residues of murine antibody variable sequences. Surface residues are defined as amino acids at least 30% of their total surface area accessible to water molecules. In the absence of structure-resolving to find surface residues of muDS6, we found 10 antibodies with the most homologous sequences from the 127 antibody structure data for alignment (fig. 12). The solvent accessibility at each Kabat position of these aligned sequences was averaged (fig. 13A and B).

The surface positions with average accessibility between 25% and 35% were subjected to a second round of analysis in which the part of the antibody that was involved in the comparison contained two identical flanking residues on either side (fig. 13A and B). After the second round of analysis, the 21 predicted surface residues of the heavy chain of muDS6 were increased to 23, and Tyr3 and Lys23 were added to the list of residues with predicted surface accessibility of greater than 30%. In most of our resurfaced antibodies, the Kabat definition of the heavy chain CDR1 was used, but for DS6 the AbM definition was inadvertently used during the calculation, so the heavy chain residue T28 was not defined as a backbone surface residue, otherwise it might be. The number of light chain surface positions decreased from 16 to 15, since the predicted surface accessibility of Ala80 decreased from 30.5% to 27.8% in the second round of analysis. The muDS6 heavy and light chain variable sequences together have 38 predicted surface accessible framework residues.

Example 8: human antibody selection

The surface position of the murine DS6 variable region was compared to the corresponding position in the human antibody sequence in the Kabat database (Johnson G, Wu TT. nucleic Acids Res. Jan 1; 29 (1): 205-6 (2001)). Surface residues from natural heavy and light chain human antibody pairs were extracted and aligned using the antibody database management software SR (Searle, 1998). Selecting the surface-pair of the variable region of the human antibody with the most identical surface residues at CDR 5Positions within are given special consideration in place of the murine DS6 antibody variable region surface residues.

Example 9: expression vectors for chimeric and humanized antibodies

The light and heavy chain paired sequences were cloned into a single mammalian expression vector. The PCR primers for the human variable sequences create restriction sites that allow the human signal sequence to be added to the pBluescriptII cloning vector. The variable sequences can then be cloned into mammalian expression plasmids with EcoRI and BsiWI or HindIII and ApaI sites for the light or heavy chain, respectively (FIG. 14). The light chain variable sequences were cloned in frame into the human Igkappa constant region and the heavy chain variable sequences were cloned into the human IgGamma constant region sequences. In the final expression plasmid, the human CMV promoter drives the expression of the light and heavy chain cDNA sequences.

Example 10: identification of residues that may negatively impact DS6 Activity

To date, for most humanization treatments, molecular models of the antibodies of interest have been established so that residues located closest to the CDRs can be identified as potential problem residues (problems). Since the number of surface reconstituted antibodies studied is increasing, historical experience is at least as effective as modeling in predicting problems, and therefore no molecular model of DS6 was established. In contrast, comparing murine DS6 surface residues to those of the antibody previously resurfaced, residues with low to high risk that could affect antibody binding activity were identified.

In available analytical antibody structures and molecular models from previous humanization work, a similar residue set was repeatedly identified as located at the 5's of the CDRWithin. Using this data, Table 1 provides the 5 that is likely to be closest to and likely to be in the CDRInner murine DS6 residues. In previous humanizations, many of these positions have also been altered, but only heavy chain position 74 once resulted in a loss of binding activity. In huC242 and huB4, murine residues were retained at this position in order to preserve the binding activity of the murine antibody. On the other hand, in humanized 6.2G5C6, this same position was changed to the corresponding human residue without loss of activity (6.2G5C6 is an anti-IGF 1-R antibody, often referred to simply as anti-C6). Although any of the residues in table 1 may be a problem in humanized antibodies, heavy chain residue P73 would be of particular interest, given previous experience with this position.

Example 11: selection of the most homologous human surface

Candidate human antibody surfaces for surface reconstruction of muDS6 were found from the Kabat antibody sequence database using SR software. The software provides an interface to search only for specific residue positions against the antibody database. To preserve natural pairing, the surface residues of the light and heavy chains are compared together. The most homologous human surfaces from the Kabat database are ranked by rank of sequence identity. The first 3 surfaces arranged by SR Kabat database software are provided in table 2. These surfaces were then compared to identify which human surfaces would require the least change to the residues identified in table 1. anti-Rh (D) antibody, 28E4(Boucher et al, 1997), requires the least number of surface residue changes (11 in total), and only three of these residues are included in the list of potentially problematic residues. Since the 28E4 antibody provided the most homologous human surface, it was all the best candidates for surface reconstituted muDS 6.

Example 12: construction of DNA sequence of humanized DS6 antibody

PCR mutagenesis was used to perform the alteration of 11 surface residues of DS 6. PCR mutagenesis was performed on murine DS6 variable region cDNA clones to construct the surface reconstituted human DS6 gene. The humanized primer set was designed to make the amino acid changes required for resurfaced DS6, as shown in table 8 below.

TABLE 8

The PCR reaction was standard except that the reaction was supplemented with 10% DMSO (50. mu.l reaction mix, 1 Xreaction buffer (ROCHE) final concentration, 2mM each dNTP, 1mM each primer, 100ng template, 5. mu.l DMSO and 0.5. mu.l Taq (ROCHE)). The reaction was carried out on an MJ Research thermocycler with the following steps: 1) 1 minute at 94 ℃; 2) 15 seconds at 94 ℃; 3) at 55 ℃ for 1 minute; 4)72 ℃ for 1 minute; 5) loop back to step #229 times; 6) the final extension step was ended at 72 ℃ for 4 minutes. The PCR product was digested with its corresponding restriction enzymes and cloned into the pBluescript cloning vector. Clones were sequenced to confirm amino acid changes.

Since changing the heavy chain residue P73 has caused problems in the past, two forms of heavy chains were established, one with human 28E4T73 and one with murine P73 retained. In both forms of humanized DS6, the other 10 surface residues were changed from murine to human 28E4 residues (table 2). The most humanized is the 1.0 form, faithful to the usual nomenclature, as it has all 11 human surface residues. The heavy chain form retaining P73 was designated the 1.2 form, and in case other forms were required, the 1.1 form was therefore retained to the form containing the most murine residues. The amino acid sequences of these two humanized forms aligned with the murine DS6 amino acid sequence are shown in fig. 15A and B. Both humanized DS6 antibody genes were cloned into antibody expression plasmids (fig. 14) for transient and stable transfection. The cDNA and amino acid sequences of the light chain variable regions of the humanized forms v1.0 and v1.2 areThe same and is shown in fig. 16. The heavy chain cDNA and amino acid sequences of the humanized forms v1.0 and v1.2 are shown in fig. 17A and B.Example 13: expression and purification of huDS6 in CHO cells and affinity determination

To determine whether the humanized DS6 form retained the binding affinity of msDS6, it was necessary to express and purify the antibody. CHO cells were transfected with the respective antibody expression plasmids. Since the transient expression level of huDS6 was very low, stable cell lines were selected.

CHODG44 cells (4.32X 10)⁶Individual cells/plate) were inoculated in 15cm plates with non-selective medium (. alpha. -MEM + nucleotides (Gibco), supplemented with 4mM L-glutamine, 50U/ml penicillin, 50. mu.g/ml streptomycin and 10% v/v FBS), and placed at 37 ℃ in 5% CO₂A humid incubator. The next day, cells were transfected with huDS6v1.0 and v1.2 expression plasmids using a modified version of the multifect transformation protocol recommended by Qiagen. The non-selective medium is aspirated from the cells. The plate was washed with 7ml of pre-warmed (37 ℃) PBS and refilled with 20ml of non-selective medium. Plasmid DNA (11. mu.g) was diluted into 800. mu.l of hybridoma SFM (Gibco). Then, 70. mu.l Polyfect (Qiagen) was added to the DNA/SFM mixture. The Polyfect mixture was then gently vortexed for a few seconds and incubated at room temperature for 10 minutes. Non-selective medium (2.7ml) was added to the mixture. This final mixture was incubated with the cells in the plate for 24 hours.

The transfection mix/medium was removed from the plate, and the cells were trypsinized and counted. Then, cells in selective medium (. alpha. -MEM-nucleotide, supplemented with 4mM L-glutamine, 50U/ml penicillin, 50. mu.g/ml streptomycin, 10% v/v FBS, 1.25mg/ml G418) were placed in 96-well plates at different concentrations (1800, 600, 200, and 67 cells/well). The cells are cultured for 2-3 weeks, and if necessary, supplemented with medium. Each well was screened for antibody production levels using a quantitative ELISA. Immulon 2HB 96-well plate was plated with goat anti-human IgG F (ab)₂Antibody coating (Jackson Immunoresearch; 1. mu.g/well in 100. mu.l of 50mM sodium carbonate buffer, pH9.6),and incubated at room temperature for 1.5h with rotation. All subsequent steps were performed at room temperature. Wells were washed twice with T-TBS (0.1% Tween-20, TBS) and blocked with 200. mu.l of blocking buffer (1% BSA, T-TBS) for 1 h. The wells were washed twice with T-TBS. In separate plates, blocking buffer was used for serial dilutions (1: 2 or 1: 3) of antibody standard, EM164(100ng/ml) and culture supernatant. These dilutions (100. mu.l) were transferred to ELISA plates and incubated for 1 h. The wells were washed three times with T-TBS and incubated for 45 min with 100. mu.l of goat anti-human IgG Fc-AP (Jackson ImmunoResearch), which had been diluted 1: 3000 with blocking buffer. After 5 washes with T-TBS, the wells were developed for 25 min using 100. mu.l of a PNPP developer (10mg/ml PNPP (disodium p-nitrophenylphosphate; Pierce), 0.1M diethanolamine pH10.3 buffer). The absorbance at 405nm was measured with an ELISA plate reader. The absorbance reading in the linear portion of the standard curve (which is the reading of the culture supernatant) is used to determine antibody levels.

The highest producer clones identified by ELISA were then expanded and frozen cell stocks were prepared. To generate sufficient amounts of antibody for purification, cells were expanded onto 15cm plates (. about.1X 10) using 30ml of selective medium⁶Cells/plate) and cultured for 1 week. The culture supernatant was collected in a 250ml conical tube, spun down in a bench top centrifuge (2000rpm, 5 minutes, 4 ℃), and then sterile filtered through a 0.2 μm filter.

To purify DS6, NaOH was added to the filtered culture supernatant to a final pH of 8.0. Hi Trap rProtein A column (Amersham) was equilibrated with 20-50 column volumes of binding buffer. The supernatant was loaded onto the column using a peristaltic pump. The column was then washed with 50 column volumes of binding buffer. The bound antibody was eluted from the column using elution buffer (100mM acetic acid, 50mM NaCl, pH3) into test tubes placed on a fraction collector. Then a neutralization buffer (2M K) was used₂HPO₄Ph10.0) and dialyzed overnight against PBS. The dialyzed antibody was filtered through a 0.2 μm syringe filter. Measuring the absorbance at 280nm to determine the final proteinMass concentration.

The affinity of purified huIgG and mudS6 was compared by flow cytometry. In the first set of experiments, direct binding to the WISH CA 6-expressing cell line was measured. As shown in figure 18A, chimeric DS6, huds6v1.0, and huDS6v 1.2 showed very similar affinities with apparent KD of 3.15nM, 3.71nM, and 4.2nM, respectively, indicating that surface reconstruction did not destroy the CDRs. These affinities were only slightly lower than the KD for muDS 6(KD ═ 1.93nM) as shown in fig. 18B. To confirm that the huDS6 form retained the affinity of mudS6, a competitive binding assay was performed. This approach has the advantage that the same detection system is used for murine and human antibodies; i.e., biotin-muDS 6/streptavidin-DTAF. In some cases, the experimentally determined apparent Kd will change only as a result of the secondary reagents used to indirectly detect binding. This was exemplified by a comparison of the apparent Kd of biotin-DS 6(Kd 2.80 nM; FIG. 18B) when using goat anti-mouse-FITC and biotin-DS 6(Kd 6.76 nM; FIG. 19A) when using streptavidin-DTAF. The results of the competitive binding assay comparing the competitive ability of muDS6, huDS6v1.0, and huDS6v 1.2 to biotin-DS 6 are shown in figure 19B. Apparent EC of muDS6, huDS6v1.0, and huDS6v 1.2₅₀12.18nM, 37.07nM and 22.64nM, respectively. These results indicate that the resurfacing of muDS6 to produce humanized DS6 caused a slight decrease in binding affinity.

Example 14: preparation of DS6-DM1 cytotoxic conjugate

DS6 antibody (8mg/ml) was modified with an 8-fold molar excess of N-succinimide-4- (2-pyridyldithio) valerate (SPP) to introduce a dithiopyridyl group. The reaction was carried out for 2h at room temperature in 95% v/v buffer A (50mM KPi, 50mM NaCl, 2mM EDTA, pH6.5) and 5% v/v DMA. The slightly swollen reaction mixture was gel filtered through a NAP or Sephadex G25 column (equilibrated in buffer a). By measuring the absorbance of the antibody at 280nm and the absorbance of DTT-released 2-mercaptopyridine (Spy) at 280 and 343nm,the extent of modification is determined. Then, at 2.5mg Ab/mL, a 1.7-fold molar excess of N over Spy was used²' -Deacetyl-N-²' - (3-mercapto-1-oxopropyl) -maytansine (L-DM1) modified DS6 was coupled. The reaction was carried out in buffer A (97% v/v) and DMA (3% v/v). The reaction was incubated overnight at room temperature for-20 h. The opaque reaction mixture was centrifuged (1162 Xg, 10 min) and the supernatant was then gel filtered through a column of NAP-25 or S300(Tandem 3, 3X 26/10 desalting column, G25 medium) equilibrated with buffer B (1XPBS pH 6.5). The precipitate was removed. The conjugate was sterile filtered using a 0.22 μm Millex-GV filter and then dialyzed against Slide-a-Lyzer in buffer B. The number of DM1 molecules attached per molecule of DS6 was determined by measuring the absorbance of the filtered material at 252nm and 280 nm. The DM1/Ab ratio was 4.36, and the stage yield of coupled DS6 was 55%. The concentration of conjugated antibody was 1.32 mg/mL. The purified conjugate, which was found to have 92% monomer, was biochemically characterized by Size Exclusion Chromatography (SEC). Analysis of DM1 in the purified conjugate showed that 99% was covalently bound to the antibody. In FIG. 20, flow cytometric binding analysis of DS6-DM1 conjugate and unmodified DS6 to Caov-3 cells indicated that conjugation of DS6 resulted in only a slight loss of affinity.

Example 15: cytotoxicity of DS6-DM1 in vitro

As a naked antibody, DS6 has been shown to have no proliferation or growth inhibitory activity in cell culture (fig. 21). However, when cells were incubated with DS6 in the presence of DM1 coupled to IgG heavy and light chains, DS6 was very effective in targeting and delivering the conjugate to the cells, resulting in indirect cytotoxicity (fig. 21). To further determine the intrinsic activity of naked DS6, Complement Dependent Cytotoxicity (CDC) assays were performed using murine and humanized DS 6. HPAC and ZR-75-1 cells (25000 cells/well) were plated in 96-well plates in 200. mu.l RHBP medium (RPMI-1640, 0.1% BSA, 20mM HEPES (pH7.2-7.4), 100U/ml penicillin and 100ug/ml streptomycin) in the presence of 5% human or rabbit serum and various dilutions of murine or humanized DS 6. Cells were incubated at 37 ℃ for 2 h. Alamar Blue (10% final concentration) reagent (Biosource) was then added to the supernatant. Cells were incubated for 5-24 hours before measuring fluorescence. Neither murine nor humanized DS6 had an effect in the Complement Dependent Cytotoxicity (CDC) assay (fig. 22), indicating that therapeutic application of DS6 requires binding of toxic effector molecules.

The cytotoxicity of maytansinoid-conjugated DS6 antibody was tested using two different assay formats with different DS6 positive cell lines. Colony generation assays were performed in which cells (1000-2500 cells/well) were plated in 6-well plates in 2ml of conjugate diluted with culture medium. At several concentrations, typically at 3X 10^-11M to 3X 10^-9M, cells were exposed to conjugate continuously and 6% CO at 37 ℃₂Incubate in a humid chamber for 5-9 days. The plates were washed with PBS and the colonies were stained with 1% w/v crystal violet/10% v/v formaldehyde/PBS solution. Unbound dye was then washed well from the wells with distilled water and the plates were allowed to dry. Colonies were counted using a leica stereozoom 4 dissecting microscope.

Plate Efficiency (PE) was calculated as the number of colonies/number of cells implanted. Survival scores were calculated as PE of treated cells/PE of untreated cells. IC was determined by plotting the fraction of cell survival versus the molar concentration of conjugate₅₀And (4) concentration. DS6-DM1 was effective at killing Caov-3 cells in a colony generation assay (FIG. 23), which estimates IC₅₀Is 800 pM. The concentration is 3X 10^-9The conjugate of M had only a slight effect on antigen-negative cells A375, 3X 10^-9M was the highest concentration of DS6-DM1 tested, indicating that the cell killing activity of the conjugate is specific for antigen expressing cells. However, while clearly sensitive to maytansine, many other DS6 positive cell lines are not particularly sensitive to immunoconjugates. All cervical cell lines (HeLa, KB and WISH) were sensitive to this conjugate, however, only a select number of ovarian and breast cell lines showed some cytotoxic effect. None of the pancreatic cell lines appeared to be affected.

In the MTT assay, cells are plated into 96-well plates at a density of 1000-5000 cells/well. Cells were plated and naked DS6 or DS6-DM1 immunoconjugate was serially diluted in 200. mu.l of medium. Samples were in triplicate. The cells and antibody/conjugate mixture were then incubated for 2-7d, at which time MTT ([3(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide)]) Assays to assess cell viability. MTT (50. mu.g/well) was added to the culture supernatant and incubated at 37 ℃ for 3-4 h. The medium was removed and MTTfumazan was dissolved in DMSO (175. mu.l/well). The absorbance at 540-545nm was measured. In the MTT cell viability assay (FIG. 24C), the immunoconjugate was effective at killing Caov-3 cells, which estimated IC₅₀It was 1.61 nM. Wells with the highest concentration of conjugate did not contain viable cells compared to the null naked antibody (fig. 21 and 24).

The results of MTT assays on other cell lines differed slightly (FIG. 24A, B and D-I). In many cases, the conjugate was unable to completely kill all cell populations (except WISH cells), although some cytotoxicity was observed. BT-20, OVCAR5 and HPAC cells are particularly resistant: in the wells with the highest conjugate concentration (32nM), more than 50% of the cells were still viable.

Example 16: in vivo conjugate antitumor Activity

To demonstrate the activity of the DS6-DM1 conjugate in vivo, human tumor xenografts were established in SCID mice. A subcutaneous model of the human cervical cancer cell line KB was established. KB cells were grown in vitro, collected and 5X 10 cells were harvested⁶The cells were injected into the right shoulder of each mouse in 100. mu.L serum-free medium and allowed to grow for 6 days with an average tumor volume of 144. + -.125 mm³At this point, drug treatment is initiated. Mice were given either PBS, 150. mu.g/kg DM1 concentration of conjugate, or 225. mu.g/kg DM1 concentration of conjugate (two mice per group) intravenously daily for 5 days. Toxicity responses were monitored daily during the treatment period. Tumor body monitoring during the course of the studyVolume (fig. 25A) and corresponding body weight (fig. 25B).

KB tumors treated with the PBS control grew rapidly with a doubling time of approximately 4 days. In contrast, both groups of mice treated with the conjugate showed complete tumor regression, 14 and 18 days after the start of treatment, respectively, for the 225 μ g/kg and 150 μ g/kg dose groups. At a dose of 150. mu.g/kg, tumors were delayed for approximately 70 days. Treatment at 225 μ g/kg resulted in a cure, as there was no evidence of tumor recurrence at study termination on day 120. As observed in fig. 25B, mice in the 150 μ g/kg group showed no weight loss, indicating that the dose was well tolerated. At higher doses, only a temporary 3% reduction in body weight occurred in the mice. The mice showed no significant signs of toxicity during the 5 day treatment period. Overall, this study showed that DS6-DM1 treatment was able to cure mice with KB xenograft tumors at non-toxic doses.

DS6-DM1 activity was further tested on a panel of subcutaneous xenograft models (see fig. 26). Tumor cell lines used to prepare xenografts showed a range of maytansine sensitivity and CA6 epitope densities in vitro (table 9 below). OVCAR5 cells and TOV-21G are ovarian tumor cell lines; HPAC is a pancreatic tumor cell line; HeLa is a cervical tumor cell line. OVCAR5 and TOV-21G cells had low surface CA6 expression; HeLa cells have moderate levels of surface CA6 expression; HPAC cells have high surface-expressed CA6 density. TOV-21G and HPAC cells are sensitive to maytansine; OVCAR5 and HeLa cells were 2-7 times less sensitive to maytansine.

TABLE 9

^*Mean maximum relative mean fluorescence

Four cell lines were cultured in vitro, harvested and 100. mu.L 1X 10 in serum-free medium⁷The cells were injected into the right shoulder of each mouse (per mouse)6 mice per model) and allowed to grow for 6 days, mean tumor volumes reached 57.6 ± 6.7 and 90.2 ± 13.4mm for the test group and control group of OVCAR5, respectively³147.1 + -29.6 and 176.2 + -18.9 mm were achieved for the HPAC test group and the control group, respectively³194.3. + -. 37.2 and 201.7. + -. 71.7mm were achieved for the test and control groups of HeLa, respectively³The test group and the control group of TOV-21G respectively reach 96.6 +/-22.8 mm and 155.6 +/-13.4 mm³At this point, drug therapy is initiated. For each model, three control mice were treated intravenously with two weekly doses of PBS and three test mice were treated intravenously with two weekly doses of conjugate (600 μ g/kg DM 1). Toxicity responses were monitored daily during treatment, tumor volume and body weight were monitored during the study. Conjugate effects for the various models are shown in fig. 26A, C, E and G, and the corresponding body weights are plotted in fig. 26B, D, F and H. OVCAR5, TOV-21G, and HPAC cell lines formed invasive tumors as observed in the PBS control of each model. The HeLa model has a lag phase of about 3 weeks before starting exponential growth. In all models, DS6-DM1 conjugate treatment resulted in complete tumor regression in all mice. For the TOV-21G, HPAC and HeLa models, mice remained tumor-free for 61 days. In the OVCAR5 model, tumors recurred approximately 45 days after tumor inoculation. Thus, DS6-DM1 treatment in this model resulted in a delay in tumor growth of approximately 34 days. Growth delay was significant because OVCAR5 cells were less sensitive to maytansine and had low expression of the CA6 epitope. In some models, CA6 epitope density was higher or the model had greater maytansine sensitivity, and tumor regression was more robust. It is important to note that only two doses were administered. It is clear that the dosage regimen used in this study was not toxic to mice, as no weight loss was observed. With additional or higher conjugate doses, healing is most likely achieved.

Human ovarian cancer is mostly a disease of the peritoneum. OVCAR5 cells grew invasively in SCID mice as an intraperitoneal model (IP), formed tumor nodules, and produced ascites in a manner similar to human disease. To demonstrate activity in the IP model, DS6-DM1 was used to treatMice with OVCAR5 IP tumors were treated (fig. 27). OVCAR5 cells were grown in vitro, harvested, and plated on 100. mu.L of 1X 10 serum-free medium⁷The individual cells were injected intraperitoneally. Tumors were allowed to grow for 6 days, at which time treatment was initiated. Mice were treated weekly for two weeks with either PBS or with DS6-DM1 conjugate at a dose of 600 μ g/kg DM1 and monitored for weight loss due to peritoneal disease. By day 28, the PBS group of mice had lost more than 20% of their body weight, which was euthanized. At day 45, the treated groups were sacrificed after more than 20% of their body weight loss. This study showed that DS6-DM1 was able to delay tumor growth in the aggressive OVCAR5 IP model, despite the fact that OVCAR5 cells were less sensitive to maytansine and had few CA6 epitopes per cell. Since the dosage regimen used does not cause visible signs of toxicity, additional or higher doses may be used to achieve further tumor delay or cure, which is likely.

Example 17: synthesis and characterization of DS6-SPP-MM1-202 Taxoid cytotoxic conjugate

DS6 was modified with an N-sulfosuccinimidyl 4-nitro-2-pyridine-pentanoate (SSNPP) linker. 10 equivalents of SSNPP in DMA were added to 90% buffer A, 50mg DS6Ab in 10% DMA. The final Ab concentration was 8 mg/ml. The reaction was stirred at room temperature for 4 hours and then purified by G25 chromatography. The degree of antibody modification was measured spectrophotometrically using the absorbance at 280nm (antibody) and 325nm (linker) and was found to have 3.82 linkers/antibody. The antibody recovery was 43.3mg, yield 87%. The DS6-nitroSPP conjugate was coupled with Taxoid MM1-202(1812 P.16). This coupling was performed on a 42mg scale in 90% buffer a, 10% DM 1. Over a period of about 20 hours, the taxoid was added in 4 equal parts of 0.43 eq/linker (per aliquot). At this point, the reaction had become noticeably cloudy. After purification at G25, the resulting conjugate, which was recovered in about 64% yield, had about 4.3 taxoids/Ab, with about 1 equivalent of unreacted linker remaining. To terminate unreacted linker, 1 equivalent of cysteine/unreacted linker was added to the conjugate while stirring overnight. Upon cysteine addition, a certain yellow hue was noted, indicating the release of thiopyridine. The reaction solution was then dialyzed against buffer B/0.01% Tween 20, followed by further dialysis against buffer B alone for several days. The final conjugate had 2.86 drugs/antibody. The recovery of antibody was 14.7mg, resulting in a total yield of 35%. The conjugate was further biochemically characterized by SEC, which was found to have 89% monomer, 10.5% dimer, and 0.5% higher molecular weight aggregates.

Flow cytometry analysis compared the binding of DS6-SPP-MM1-202 taxoid and DS6 antibodies on HeLa cells, and the results are shown in FIG. 28. This result shows that DS6 retains binding activity when it is coupled to a taxane (taxane).

Sequence listing

<110> Issudacon corporation (ImmunoGen, Inc.)

G-pei En (PAYNE, Gillian)

P.dun (CHUN, Philip)

D, tavarez (TAVARES, Daniel)

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<151>2003-07-21

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cag ggc aag gcc aca ttg act gca gac cca tcc tcc agc aca gcc tac 240

Gln Gly Lys Ala Thr Leu Thr Ala Asp Pro Ser Ser Ser Thr Ala Tyr

65 70 75 80

atg cag atc agc agc ctg aca tct gaa gac tct gcg gtc tat ttc tgt 288

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala ValTyr Phe Cys

85 90 95

gca aga gga gat tcg gtc ccg ttt gct tac tgg ggc caa ggg act ctt 336

Ala Arg Gly Asp Ser Val Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

gtc act gtc tct gcc 351

Val Thr Val Ser Ala

115

<210>12

<211>15

<212>PRT

<213> mouse

<400>12

Gln Val Thr Ala Ile Pro Lys Pro Gly Gly Ala Ser Arg Glu Lys

1 5 10 15

<210>13

<211>15

<212>PRT

<213> human (Homo sapiens)

<400>13

Glu Val Thr Ala Thr Pro Arg Pro Gly Gly Ala Ser Ser Glu Lys

1 5 10 15

<210>14

<211>15

<212>PRT

<213> human

<400>14

Glu Val Thr Gly Thr Pro Arg Pro Gly Gly Asp Ser Arg Glu Lys

1 5 10 15

<210>15

<211>15

<212>PRT

<213> human

<400>15

Glu Val Thr Gly Thr Pro Arg Pro Gly Gly Asp Ser Arg Glu Lys

1 5 10 15

<210>16

<211>23

<212>PRT

<213> mouse

<400>16

Gln Tyr Gln Ala Leu Arg Ser Lys Lys Pro Gly Gln Gln Lys Lys Gly

1 5 10 15

Pro Ser Ser Ser Glu Gln Ser

20

<210>17

<211>23

<212>PRT

<213> human

<400>17

Gln Gln Val Ala Val Lys Pro Lys Lys Pro Gly Gln Gln Lys Gln Gly

1 5 10 15

Thr Ser Ser Ser Glu Gln Ser

20

<210>18

<211>22

<212>PRT

<213> human

<400>18

Gln Val Ala Val Lys Pro Lys Lys Pro Gly Gln Gln Lys Gln Gly Glu

1 5 10 15

Ser Ser Ser Glu Gln Ser

20

<210>19

<211>22

<212>PRT

<213> human

<400>19

Gln Val Ala Val Lys Pro Lys Lys Pro Gly Gln Gln Lys Gln Gly Glu

1 5 10 15

Ser Ser Ser Glu Gln Ser

20

<210>20

<211>10

<212>PRT

<213> mouse

<400>20

Gly Tyr Thr Phe Thr Ser Tyr Asn Met His

1 5 10

<210>21

<211>10

<212>PRT

<213> mouse

<400>21

Tyr Ile Tyr Pro Gly Asn Gly Ala Thr Asn

1 5 10

<210>22

<211>8

<212>PRT

<213> mouse

<400>22

Gly Asp Ser Val Pro Phe Ala Tyr

1 5

<210>23

<211>95

<212>PRT

<213> mouse

<400>23

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Pro

85 90 95

<210>24

<211>98

<212>PRT

<213> mouse

<400>24

Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg

<210>25

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>25

tatagagctc aagcttggat ggtgggaaga tggatacagt tggtgc 46

<210>26

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>26

ggaggatcca tagacagatg ggggtgtcgt tttggc 36

<210>27

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>27

gggagctcga yattgtgmts acmcarwctm ca 32

<210>28

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<220>

<221>misc_feature

<222>(18)..(18)

<223> n is a, c, g or t

<400>28

cttccggaat tcsargtnma gctgsagsag tc 32

<210>29

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<220>

<221>misc_feature

<222>(18)..(18)

<223> n is a, c, g or t

<400>29

cttccggaat tcsargtnma gctgsagsag tcwgg 35

<210>30

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> degenerate primers

<400>30

ttttgaattc aataactaca ggtgtccact 30

<210>31

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> degenerate primers

<400>31

ttttgagctc cagattttca gcttcctgct 30

<210>32

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>32

cgatgggccc ttggtggagg ctgcagagac agtgaccaga 40

<210>33

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>33

ttttcgtacg tttcagctcc agcttggt 28

<210>34

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>34

caggtgtaca ctcccaggct tatctccagc agtct 35

<210>35

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>35

cgatgggccc ttggtggagg cggcagagac agtgaccaga 40

<210>36

<211>56

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>36

caggtgtaca ctccgagatt gttctcaccc agtctccagc aaccatgtct gcatct 56

<210>37

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>37

ggcactgcag gttatggtga ccctctcccc tggaga 36

<210>38

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>38

caatcagcag catggaggct gaaga 25

<210>39

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>39

gcctccatgc tgctgattgt gaga 24

<210>40

<211>67

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>40

caggtgtaca ctcccaggct cagctcgtgc agtctggggc tgaggtggtg aagcccgggg 60

cctcagt 67

<210>41

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>41

ttgactgcag acacatcctc cagcaca 27

<210>42

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>42

gtgtctgcag tcaatgtggc cttgccctgg aacttctgat 40

<210>43

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400>43

cgatgggccc ttggtggagg cggcagagac agtgacaaga 40

<210>44

<211>107

<212>PRT

<213> mouse

<400>44

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Phe Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Ser Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Ile Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210>45

<211>107

<212>PRT

<213> mouse

<220>

<221>misc_feature

<222>(1)..(1)

<223> Xaa can be any naturally occurring amino acid

<400>45

Xaa Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Asn Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Phe Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Thr Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Gly Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Gly Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210>46

<211>107

<212>PRT

<213> mouse

<400>46

Asp Ile Gln Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Ser Thr Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210>47

<211>108

<212>PRT

<213> mouse

<400>47

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Tyr Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile

85 90 95

Thr Phe Gly Val Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210>48

<211>105

<212>PRT

<213> mouse

<400>48

Gln Ile Val Ser Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Arg Ser Tyr Met

20 25 30

Gln Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Thr Phe Gly

85 90 95

Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210>49

<211>107

<212>PRT

<213> mouse

<400>49

Gln Thr Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210>50

<211>107

<212>PRT

<213> mouse

<400>50

Gln Ser Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Ile Met Thr Cys Ser Pro Ser Ser Ser Val Ser Tyr Met

20 25 30

Gln Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Gly

50 55 60

Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile Ser Gly Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210>51

<211>109

<212>PRT

<213> mouse

<400>51

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Ala Cys Arg Ala Ser Ser Ser Val Ser Ser Thr

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210>52

<211>110

<212>PRT

<213> mouse

<400>52

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala

100 105 110

<210>53

<211>109

<212>PRT

<213> mouse

<400>53

Asp Ile Gln Leu Thr Gln Ser Pro Ala Phe Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser

20 25 30

Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp

35 40 45

Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Tyr Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210>54

<211>117

<212>PRT

<213> mouse

<400>54

Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Lys Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210>55

<211>122

<212>PRT

<213> mouse

<400>55

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ile Phe Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Gly Ser Tyr Arg Tyr Asp Gly Gly Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>56

<211>119

<212>PRT

<213> mouse

<400>56

Gln Gly Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Phe

20 25 30

Trp Val Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Asn Lys Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Tyr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Asn Tyr Pro Tyr ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210>57

<211>120

<212>PRT

<213> mouse

<400>57

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>58

<211>121

<212>PRT

<213> mouse

<400>58

Glu Val Gln Leu Gln Gln Ser Gly Val Glu Leu Val Arg Ala Gly Ser

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn

20 25 30

Gly Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Asn Asn Pro Gly Asn Gly Tyr Ile Ala Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Glu Tyr Tyr Gly Gly Ser Tyr Lys Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>59

<211>120

<212>PRT

<213> mouse

<400>59

Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Asn Trp Met Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Lys Thr Thr Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ala

115 120

<210>60

<211>120

<212>PRT

<213> mouse

<400>60

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Phe

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Gly Thr His Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly His Ser Tyr Tyr Phe Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>61

<211>124

<212>PRT

<213> mouse

<400>61

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ala Gly Ser

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Val Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Gly Lys Gly Tyr Leu Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ala Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Phe Tyr Gly Gly Ser Asp Leu Ala Val Tyr Tyr Phe Asp

100 105 110

Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>62

<211>116

<212>PRT

<213> mouse

<400>62

Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Thr Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Gly Asn Tyr Gly Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210>63

<211>116

<212>PRT

<213> mouse

<400>63

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Asp Asp Asn Tyr Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val

115

Claims (58)

1. A humanized antibody or epitope-binding fragment thereof comprising at least one complementarity-determining region having a sequence set forth in SEQ ID NOS: 1.2 and 3 and 4-6.

2. A humanized antibody or epitope-binding fragment thereof comprising at least one heavy chain variable region or fragment thereof and at least one light chain variable region or fragment thereof, wherein said at least one heavy chain variable region or fragment thereof comprises three sequence complementarity determining regions having sequences set forth in SEQ ID NOS: 1.2 and 3;

and wherein the at least one light chain variable region or fragment thereof comprises three sequence complementarity determining regions having sequences set forth in SEQ ID NOS: 4-6.

3. The antibody or epitope-binding fragment thereof of claim 2, wherein the light chain variable region or fragment thereof has the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8.

4. The antibody or epitope-binding fragment thereof of claim 2, wherein the heavy chain variable region or fragment thereof has the sequence set forth in SEQ ID NO: 9. SEQ ID NO: 10 or SEQ ID NO: shown at 11.

5. The antibody or epitope-binding fragment thereof of claim 2, wherein the light chain variable region or fragment thereof has the sequence set forth in SEQ ID NO: 8, and the sequence of the heavy chain variable region or the fragment thereof is shown as SEQ ID NO: shown at 11.

6. A polynucleotide encoding the antibody or epitope-binding fragment thereof of any one of claims 1-5.

7. A polynucleotide encoding the light or heavy chain of the antibody or epitope-binding fragment thereof of any one of claims 1-5.

8. A vector comprising the polynucleotide of claim 6.

9. A vector comprising the polynucleotide of claim 7.

10. The vector of claim 8, wherein the vector is an expression vector that expresses the antibody or epitope-binding fragment thereof.

11. The vector of claim 9, wherein the vector is an expression vector that expresses the antibody or epitope-binding fragment thereof.

12. A cytotoxic conjugate comprising the antibody or epitope-binding fragment thereof of any one of claims 1-5 and a cytotoxic agent, wherein the antibody or epitope-binding fragment thereof binds to a CA6 glycotope.

13. The cytotoxic conjugate of claim 12, wherein the antibody or epitope-binding fragment thereof and the cytotoxic agent are covalently linked.

14. The cytotoxic conjugate of claim 12, wherein the antibody or epitope-binding fragment thereof and the cytotoxic agent are covalently linked by a PEG linking group.

15. The cytotoxic conjugate of claim 12, wherein the antibody or epitope-binding fragment thereof and the cytotoxic agent are covalently linked through a thiol or disulfide functional group of the cytotoxic agent.

16. The cytotoxic conjugate of claim 12, wherein the antibody or epitope-binding fragment thereof is selected from the group consisting of: a polyclonal antibody; a monoclonal antibody; an antibody fragment; resurfaced antibodies; improved antibodies; and antibodies including interferon, lymphokine, hormone, growth factor, transferrin, or vitamin.

17. The cytotoxic conjugate of claim 16, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab 'fragment, F (ab')₂Fragments, Fd fragments, single chain Fvs fragments, single chain antibodies, disulfide linked Fvs fragments, and fragments comprising a VL or VH domain.

18. The cytotoxic conjugate of claim 12, wherein the antibody or epitope-binding fragment thereof is an anti-CA 6 monoclonal antibody or epitope-binding fragment thereof.

19. The cytotoxic conjugate of claim 12, wherein the cytotoxic agent is selected from the group consisting of a maytansinoid compound, a taxane compound, a CC-1065 compound, a dolastatin compound, a daunorubicin compound, and a doxorubicin compound.

20. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is maytansine N of formula (I)^2’-deacetyl-N^2’- (3-mercapto-1-oxopropyl) -maytansine:

21. the cytotoxic conjugate of claim 19, wherein the cytotoxic agent is maytansine N of formula (II)^2’-deacetyl-N^2’- (4-methyl-4-mercapto-1-oxopentyl) -maytansine:

22. the cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine of formula (III):

wherein:

y' represents

(CR₇CR₈)_l(CR₉＝CR₁₀)_pC＝C_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C＝C)_sB_t(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocycloaryl or heterocycloalkyl, and R₂May be H;

A. b, D is cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl or heteroaryl or heterocycloalkyl;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not simultaneously 0; and

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group.

23. The cytotoxic conjugate of claim 22, wherein R₁Is H, R₂Is methyl and Z is H.

24. The cytotoxic conjugate of claim 22, wherein R₁And R₂Is methyl and Z is H.

25. The cytotoxic conjugate of claim 22, wherein R₁Is H, R₂Is methyl, Z is-SCH₃。

26. The cytotoxic conjugate of claim 22, wherein R₁And R₂Is methyl, Z is-SCH₃。

27. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine selected from the group consisting of formulas (IV-L), (IV-D), and (IV-D, L):

wherein:

y represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic alkyl group, and further R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl groups having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl groups having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic alkyl group;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0;

z is H, SR or-COR, wherein R is a linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, a cycloalkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aryl or heterocycloalkyl group; and

may represents maytansinoids having a side chain at the C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation.

28. The cytotoxic conjugate of claim 27, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0, and Z is H.

29. The cytotoxic conjugate of claim 27, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0 and Z is H.

30. The cytotoxic conjugate of claim 27, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0 and Z is-SCH₃。

31. The cytotoxic conjugate of claim 27, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0 and Z is-SCH₃。

32. The cytotoxic conjugate of claim 27, wherein the cytotoxic agent is represented by formula (IV-L).

33. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine of formula (V):

wherein:

y represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl, and R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0; and

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group.

34. The cytotoxic conjugate of claim 33, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0 and Z is H.

35. According toThe cytotoxic conjugate of claim 33, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0 and Z is H.

36. The cytotoxic conjugate of claim 33, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H, l and m are each 1, n is 0 and Z is-SCH₃。

37. The cytotoxic conjugate of claim 33, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Each is H, l and m are 1, n is 0 and Z is-SCH₃。

38. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine selected from the group consisting of formulas (VI-L), (VI-D), and (VI-D, L):

wherein:

Y₂represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocycloaryl or heterocycloalkyl, and R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0;

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group; and

may is a maytansinoid.

39. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine of formula (VII):

wherein:

Y₂' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C＝C)_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C＝C)_sB_t(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cycloalkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl, and, in addition, R₂May be H;

A. b and D are each independently cycloalkyl or cycloalkenyl having from 3 to 10 carbon atoms, simple or substituted aryl, or heteroaryl or heterocycloalkyl;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not simultaneously zero; and

Z₂is SR or-COR, where R is a linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group.

40. The cytotoxic conjugate of claim 39, wherein R₁Is H, R₂Is methyl.

41. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine of formula (VIII):

wherein:

Y₁' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C＝C)_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C＝C)_sB_t(CR₃CR₄)_nCR₁R₂S-，

Wherein:

A. b and D are each independently cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl, or heteroaryl or heterocycloalkyl;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups; and

l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not simultaneously zero.

42. The cytotoxic conjugate of claim 41, wherein R₁Is H, R₂Is methyl.

43. The cytotoxic conjugate of claim 41, wherein R₁And R₂Is methyl.

44. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine selected from the group consisting of formulas (IX-L), (IX-D), and (IX-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl radicals having 1 to 10 carbon atomsA radical, a branched or cyclic alkyl or alkenyl radical having from 3 to 10 carbon atoms, a phenyl radical, a substituted phenyl radical, or a heteroaromatic or heterocycloalkyl radical, and in addition R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups;

l, m and n are each independently an integer of 1 to 5, and further, n may be 0; and

may represents maytansinol, which has a side chain at a C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation.

45. The cytotoxic conjugate of claim 44, wherein R₁Is H and R₂Is methyl, or R₁And R₂Is methyl.

46. The cytotoxic conjugate of claim 44, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H; l and m are each 1; n is 0.

47. The cytotoxic conjugate of claim 44, wherein R₁And R₂Is methyl: r₅、R₆、R₇And R₈Each is H; l and m are 1; n is 0.

48. The cytotoxic conjugate of claim 45, wherein the maytansinoid is represented by formula (IX-L).

49. The cytotoxic conjugate of claim 46, wherein the maytansinoid is represented by formula (IX-L).

50. The cytotoxic conjugate of claim 47, wherein the maytansinoid is represented by formula (IX-L).

51. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a maytansine of formula (X):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-，

Wherein:

R₁and R₂Each independently is CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl, and R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, CH₃、C₂H₅Linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl groups; and

l, m and n are each independently an integer of 1 to 5, and further, n may be 0.

52. The cytotoxic conjugate of claim 51, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Each is H; l and m are each 1; n is 0.

53. The cytotoxic conjugate of claim 51, wherein R is₁And R₂Is methyl; r₅、R₆、R₇And R₈Each is H; l and m are 1; n is 0.

54. The cytotoxic conjugate of claim 12, wherein the antibody or epitope-binding fragment thereof comprises a light chain variable region comprising a sequence set forth in SEQ ID NO: 8 and the sequence thereof is shown as SEQ ID NO: 11, wherein the cytotoxic agent is N^2’-deacetyl-N^2’- (3-mercapto-1-oxopropyl) -maytansine or N^2’-deacetyl-N^2’- (4-methyl-4-mercapto-1-oxopentyl) -maytansine.

55. The cytotoxic conjugate of claim 19, wherein the cytotoxic agent is a taxane of the formula:

56. a therapeutic composition comprising the cytotoxic conjugate of claim 12 and a pharmaceutically acceptable carrier or excipient.

57. The therapeutic composition of claim 56, wherein the cytotoxic conjugate comprises a peptide having a sequence set forth in SEQ ID NO: 8 and the sequence thereof is shown as SEQ ID NO: 11, and N^2’-deacetyl-N^2’- (3-mercapto-1-oxopropyl) -maytansine or N^2’-deacetyl-N^2’- (4-methyl-4-mercapto-1-oxopentyl) -maytansine as cytotoxic agent.

58. The therapeutic composition of claim 56, wherein the cytotoxic conjugate comprises a peptide having a sequence set forth in SEQ ID NO: 8 and the sequence thereof is shown as SEQ ID NO: 11, and a taxane as a cytotoxic agent.