MX2008002607A - A ca6 antigen-specific cytotoxic conjugate and methods of using the same. - Google Patents

Abstract

Cytotoxic conjugates comprising a cell binding agent and a cytotoxic agent, therapeutic compositions comprising the conjugate, methods for using the conjugates in the inhibition of cell growth and the treatment of disease, and a kit comprising the cytotoxic conjugate are disclosed are all embodiments of the invention. In particular, the cell binding agent is a monoclonal antibody, and epitope-binding fragments thereof, that recognizes and binds the CA6 glycotope. The present invention is also directed to humanized or resurfaced versions of DS6, an anti-CA6 murine monoclonal antibody, and epitope-binding fragments thereof.

Description

AN ANTIGEN-SPECIFIC CA6 CITOTOXIC CONJUGATE AND METHODS TO USE THE SAME FIELD OF THE INVENTION The present invention is directed to murine anti-CA6 glycoprotein monoclonal antibody, and humanized or coated versions thereof. The present invention is also directed to epitope binding fragments of the anti-CA6 glycoprotein monoclonal antibody, as well as to epitope binding fragments of humanized or coated versions of the anti-CA6 glycoprotein monoclonal antibody. The present invention is further directed to cytotoxic conjugates comprising a cell-binding agent and a cytotoxic agent, therapeutic compositions comprising the conjugate, methods for using the conjugates in the inhibition of cell growth and treatment of the disease, and an equipment that comprises the cytotoxic conjugate. In particular, the cell binding agent is a monoclonal antibody, or epitope-binding fragment thereof, that recognizes and binds the CA6 glycopeto or a humanized or coated version thereof.

BACKGROUND OF THE INVENTION There have been numerous attempts to develop agents anti-carcinogenic therapeutics that specifically destroy target cancer cells without harming nearby non-carcinogenic cells and tissue. Such therapeutic agents have the potential to greatly improve the treatment of cancer in human patients. A promising approach has been to bind cell-binding agents, such as monoclonal antibodies, with 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), Diener et al, in Antibody mediated delivery systems 1-23 (J. Rodwell, ed., 1988), Pietersz 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 selection of the cell-binding agent, these cytotoxic conjugates can be designed to recognize and bind only to types. of cancer cells, based on the expression profile of the molecules expressed on the surface of said cells Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been used in said cytotoxic conjugates , linked to a variety of murine monoclonal antibodies. In some cases, the drug molecules were linked to the antibody molecules through an intermediate carrier molecule such as serum albumin (Garnett et al, 46 Cancer Res. 2407-2412 (1986); Ohkawa et al 23 Cancer Immunol. Immunother. 81-86 (1986); Endo et al, 47 Cancer 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, 85 Proc. Nati. Acad. Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al, 73 J. Nati. Inst. 721-729 (1984), Kato et al 27 J. Med. Chem. 1602-1607 (1984), Tsukada et al, 52 Br. J. Cancer 111-116 (1985)). As an example of a specific coinjugate that has shown some promise, there is the C242 antibody conjugate, directed against CanAg, an antigen expressed in colorectal and pancreatic tumors, and the maytansine derivative DMI (Liu et al., Proc Nati Acad Sci USA , 93: 8618-8623 (1996)). The in vitro evaluation of this conjugate indicated that its binding affinity towards CanAg expressed on the cell surface was eluded with an apparent Kd value of 3 x 1? '11 M, and its cytotoxic potential for CanAg-positive cells was elevated with an IC50. of 6 x 10 ° 11 M. This cytotoxicity was antigen-dependent since it was blocked by an excess of unconjugated antibody and, since the antigen-negative cells were 100-fold less sensitive to the conjugate. Other examples of antibody-DMI conjugates with high affinity both to target cells and to high antigen-selective cytotoxicity include those of huN901, a humanized version of antibody to human CD56; huMy9-6, a humanized version of antibody against human CD33; huC242, a humanized version of antibody against the CanAg Mucl epitope; huJ591, a deimmunized antibody against PSMA; trastuzumab, a humanized antibody against Her2 / neu; and bivatuzumab, a humanized antibody against CD44v6. The development of additional cytotoxic conjugates that specifically recognize specific types of cancer cells will be important in the continuous improvement of the methods used to treat patients with cancer. For that purpose, the present invention is directed to the development of antibodies that recognize and bind molecules / receptors expressed on the surface of cancer cells, and to the development of novel cytotoxic conjugates comprising cell binding agents, such as antibodies, and cytotoxic agents that they target specific molecules / receptors expressed on the surface of cancer cells. More specifically, the present invention is directed to the characterization of a novel CA6 sialogliquetope in the Mucl mucin receptor expressed by cancer cells, and to the delivery of antibodies, preferably humanized antibodies, which recognize the CA6 sialogliquetope of the Mucl mucin receptor and It can be used to inhibit the growth of a cell that expresses the CA6 glycopeto in the context of a cytotoxic agent.

BRIEF DESCRIPTION OF THE INVENTION The present invention includes antibodies that specifically recognize and bind a novel CA6 sialogliquetope of the Mucl mucin receptor, or an epitope binding fragment thereof. In another embodiment, the present invention includes a humanized antibody, or an epitope-binding fragment thereof, that recognizes the novel CA6 sialogliquetope ("the CA6 glycopetope") of the Mucl mucin receptor. [11] In preferred embodiments, the present invention includes murine anti-CA6 monoclonal antibodies ("the DS6 antibody"), and coated or humanized versions of the DS6 antibody wherein those exposed to the surface of the antibody, or their binding fragments of the antibody. epitope, are replaced in both light and heavy chains to more closely resemble known human antibody surfaces. The humanized antibodies and epitope-binding fragments thereof of the present invention have improved properties since they are much less immunogenic (or completely non-immunogenic) in human patients to whom they are administered than the completely murine versions. Therefore, the humanized DS6 antibodies and epitope-binding fragments thereof of the present invention specifically recognize a novel sialoglicatope at the Mucl mucin receptor, ie the CA6 glycoprotein, as long as it is not immunogenic for a human being. human. Antibodies Humanized and epitope-binding fragments thereof can be conjugated to a drug, such as a maitansionoid, to form a prodrug that has specific cytotoxicity to the cells expressing antigen by directing the drug to the Mucl CA6 sialogliquetope. Cytotoxic conjugates comprising said antibodies and some highly toxic drugs (eg, maytansinoids, taxanes, and analogs CC-1065) may therefore be used as a therapeutic for the treatment of tumors, such as breast and ovarian tumors. The humanized versions of the DS6 antibody of the present invention are fully characterized herein with respect to their respective amino acid sequences of both light and heavy chain variable regions, the DNA sequences of the genes for the light and heavy chain variable regions, the identification of the CDRs, the identification of their surface amino acids, and the description of a means for their expression in recombinant form. In one embodiment, a humanized DS6 antibody or an epitope-binding fragment thereof having a heavy chain including CDRs having amino acid sequences represented by NOS SEQ ID: 1-3: SYNMH (NO ID SEC: 1) is provided. , YIYPGNGATNYNQKFKG (NO SEC ID: 2), GDSVPFAY (NO ID SEC: 3), and having a light chain comprising CDRs that have amino acid sequences represented by NOS SEC ID: 4-6: SAHSSVSFMH (NO SEC ID: 4), STSSLAS (NO SEC ID: 5), QQRSSFPLT (NO SEC ID: 6), Humanized DS6 antibodies and binding fragments are also provided of epitope thereof having a light chain variable region possessing an amino acid sequence that shares at least 90% sequence identity with an amino acid sequence represented by SEQ ID N0: 7 or NO SEQ ID: 8: QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLW IYSTSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLT FGAGTKLELKR (SEQ ID N0: 7) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKL WIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPL TFGAGTKLELKR (SEQ ID NO: 8) Similarly, humanized DS6 antibodies and epitope-binding fragments thereof having a light chain variable region possessing an amino acid sequence that shares at least 90% identity are provided. sequence with an amino acid sequence represented by SEQ ID NO: 9, NO SEC ID: 10, or NO SEQ ID: 11: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEWIGYIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 9) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE WIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (NO ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE WIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (NO ID NO: 11) In another embodiment, the DS6 humanized antibodies and binding fragments epitope thereof are provided for a variable region humanized light chain or coated that has an amino acid sequence that corresponds to NO ID SEC: 8 EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKL WIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPL TFG AGTKLELKR. (NO SEC ID: 8). Similarly, humanized DS6 antibodies and epitope-binding fragments thereof are provided to have a humanized or coated heavy chain region possessing an amino acid sequence corresponding to NO SEQ ID NO: 10 or NO SEQ ID NO: 11, respectively: QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA. (NO SEC ID: 10) QAQLVQSGAEWKPGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA. (NO SEC ID: 11) The humanized DS6 antibodies and epitope-binding fragments thereof of the present invention may also include substitution at light and / or heavy chain amino acid residues at one or more positions defined by the residues marked with an asterisk in Table 1 representing surface residues of murine structure found in 5 Angstroms of a CDR that requires change for a human waste. For example, the first Q amino acid residue in the murine sequence (NO SEQ ID: 7) has been replaced by E (NO SEQ ID: 8) to humanize the antibody. However, due to the proximity of this residue to a CDR, a backmowing for the murine Q residue may be required in order to maintain the affinity of the antibody.

Table 1 This is shown additionally in Table 2 where the surface residues of the variable muDSo region are shown to be aligned with the three human variable region surface residues plus homologues. The amino acid residues in Table 1 correspond to the amino acid residues underlined in Table 2. Table 2 [20] The present invention further provides cytotoxic conjugates comprising (1) a cell binding agent that recognizes and binds the glycopept CA6, and (2) a cytotoxic agent. In the cytotoxic conjugates, the cell binding agent has high affinity for the glycoprote CA6 and the cytotoxic agent has a high degree of cytotoxicity for cells expressing the CA6 glycoptotope, so that the cytotoxic conjugates of the present invention form eliminating agents effective. In a preferred embodiment, the cell binding agent is an anti-CA6 antibody or an epitope-binding fragment thereof, more preferably a humanized anti-CA6 antibody or an epitope-binding fragment thereof, wherein a cytotoxic agent is covalently linked, directly or through a separable or non-separable linker, to the antibody or epitope binding fragment. of the same. In more preferred embodiments, the cell binding agent is the humanized DS6 antibody or an epitope-binding fragment thereof, and the cytotoxic agent is a taxol, a maitansionoid, .CC-1065 or a CC-1065 analog. In preferred embodiments of the invention, the cell binding agent is a humanized anti-CA6 antibody and the cytotoxic agent is a cytotoxic drug such as a maitansionoid or a taxane. More preferably, the cell binding agent is the humanized anti-CA6 antibody DS6 and the cytotoxic agent is an aitansine compound, such as DM1 or DM4. The present invention also includes a method for inhibiting the growth of a cell expressing the CA6 glycopept. In preferred embodiments, the method for inhibiting the growth of the cell expressing the CA6 glycopeptum occurs in vivo and results in cell death, although in vitro and ex vivo applications are also included. The present invention also provides a therapeutic composition comprising the cytotoxic conjugate, and a pharmaceutically acceptable carrier or excipient. The present invention further includes a method for treating a subject having cancer utilizing the therapeutic composition. In Preferred embodiments, the cytotoxic conjugate comprises an anti-CA6 antibody and a cytotoxic agent. In more preferred embodiments, the cytotoxic conjugate comprises a humanized D6-conjugated DMI antibody, humanized DS6 antibody-DM4 or a humanized-conjugated taxane DS6 antibody, and the conjugate is administered together with a pharmaceutically acceptable carrier or excipient. The present invention also includes a kit comprising an anti-CA6-conjugated antibody of cytotoxic agent and instructions for use. In preferred modes, the anti-CA6 antibody is the humanized DS6 antibody, the cytotoxic agent is a maytansine compound, such as DMI or DM4, or a taxane, and the instructions are to use the conjugates in the treatment of a subject having cancer. . The kit can also include the components necessary for the preparation of a pharmaceutically acceptable formulation, such as a diluent if the conjugate is in a lyophilized state or in a concentrated form and for the administration of the formulation. The present invention also includes antibody derivatives that specifically bind and recognize the CA6 glycopept. In preferred embodiments, the antibody derivatives are prepared by coating or humanizing antibodies that bind the CA6 glycopeto, wherein the derivatives have decreased immunogenicity towards the recipient. The present invention also provides antibodies humanized or fragments thereof that are additionally 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. A method for diagnosis is also provided in which the labeled humanized antibodies or epitope binding fragments thereof are administered to a subject presumed to have a cancer, and the distribution of the tag within the subject's body is measured or monitored. . The present invention also provides methods for the treatment of a subject having a cancer by administration of a humanized antibody conjugate of the present invention, either alone or in combination with other cytotoxic or therapeutic agents. The cancer may be one or more, for example, of breast cancer, colon cancer, ovarian carcinoma, endometrial cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial carcinoma, pancreatic cancer, a sarcoma or a carcinoma in which expresses CA6 or another cancer not yet determined in which the CA6 glycoptol is predominantly expressed. Unless stated otherwise, all references and patents cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of studies performed to determine the ability of the DS6 antibody to bind the surface of selected cancer cell lines. The fluorescence of cell lines incubated the primary antibody DS6 and secondary antibodies of FITC anti-mouse conjugate IgG (H + L) was measured through flow cytometry. The DS6 antibody bound Caov-3 cells (Figure 1A) and T-47D (Figure 1B) with an apparent Kd of 1848 nM and 2586 nM respectively. The antigen-negative cell lines, SK-OV-3 (Figure 1C) and Colo205 (Figure 1D) showed no specific antigen binding. Figure 2 shows the results of the expression epitope point staining analysis. Cell lysates Caov-3 (Figure 2A &Figure 2B), SKMEL28 (Figure 2C), and Colo205 (Figure 2D) were individually stained on nitrocellulose membranes and then incubated individually with pronase, proteinase K, neuraminidase or periodic acid. The membranes were then immunostained with the DS6 antibody (Figure 2A), the CMI antibody (Figure 2B), the R24 antibody (Figure 2C), or the C242 antibody (Figure 2D). Figure 3 shows the results of a spot staining analysis of DS6 antigen expression. Caov-3 cell lysates were stained individually on PVDF membranes and then incubated in the presence of trifluoromethanesulfonic acid (TFMSA). The membranes were immunostained with the CMI antibody (1 & 2) or the DS6 antibody (3 & 4). Figure 4 shows the results of the glycopept analysis of the DS6 antigen. Caov-3 lysates pretreated with N-glycanase ("N-gly"), O-glycanase ("O-gly"), and / or sialidase ("S") were stained on nitrocellulose and then immunostained with the DS6 antibody or the CMI antibody (Muc-1 VNTR). Figure 5 shows the results of Western blot analysis of the DS6 antigen. The cell lysates were immunoprecipitated ("IP") and immunostained with the DS6 antibody. The antigen corresponds to a protein band > 250 kDa observed in Caov-3 antigen-positive cells (Figure 5A and Figure 5B) and T47D (Figure 5C). the antigen-negative cell lines SK-OV-3 (Figure 5D) and Colo205 (Figure 5E) do not exhibit this band. After immunoprecipitation, the Protein G beds of the Caov-3 cell lysates were incubated with (Figure 5A) neuraminidase ("N") or (Figure 5B) periodic acid ("PA"). The antibody lysate controls ("a"), pre-IP ("Lys") and post-IP flow ("FT") were operated on the same gel. Immunoprecipitates Caov-3 were also incubated with N-glycanase ("N-gly"), O-glycanase ("O-gly"), and / or sialidase ("S") (see Figure 5F), where the Staining was analyzed alternatively with biotinylated DS-6 and strepavidin-HRP. Figure 6 shows the results of immunoprecipitations and / or immunoblots of the DS6 antibody and the CMI antibody antibody in cell lysates Caov-3 (Figure 6A) and HeLa (Figure 6B). The overlap of Western CMI and DS6 staining signals means that the DS6 antigen is in the Mucl protein. In the HeLa lysates the double Mucl results from the Mucl expression directed by different alleles that differ in their number of tandem repeats. Figure 7 shows an Elisa interspersed design of antibody DS6 (Figure 7A) and a standard curve (Figure 7B). The standard curve was generated using known concentrations of commercially available CAI 5-3 standards (where unit 1 CAI 5-3 = unit 1 DS6). Figure 8 shows standard quantitative ELISA curves. The standard curves of antibody signal detection (streptavidin-HRP / biotin-DS6) (Figure 8C) were determined using known concentrations of biotin-DS6 either captured by goat anti-mouse IgG on plates (Figure 8A) or linked directly on the ELISA plate (Figure 8B). Figure 9 shows the cDNA and amino acid sequences of the light chain variable region (Figure 9A) and heavy chain (Figure 9B) for the murine DS6 antibody. The three CDRs in each sequence are underlined (Kabat definitions). Figure 10 shows the light (Figure 10A) and heavy (Figure 10B) CDRs of the murine DS6 antibody determined by the Kabat definitions. The AbM modeling software produces a slightly different definition for the heavy chain CDRs (Figure 10C).

Figure 11 shows the amino acid sequences of light chain ("muDSOLC") (residues 1 -95 of SEQ ID NO: 7) and of heavy chain ("muDS6HC") (residues 1-98 of NO SEQ ID: 9) for the murine DS6 antibody aligned with the germline sequences for the genes lgV? ap4 (NO SEC ID: 23) and IgVh J558.41 (NO SEC ID: 24). The gray color indicates divergence of sequence. Figure 12 shows the ten most homologous light chain and heavy chain antibody sequences for murine ("muDS6LC") and heavy chain ("muDS6HC") DS6 (muDS6) sequences that have resolved the archives of structure in the Brookhaven database. The sequences are aligned in order from most to least homologous. Figure 13 shows data and calculations of surface access capacity to predict which structure residues of the light chain variable region of the murine DS6 antibody are surface accessible. The positions with 25-35% average surface access capacity are marked (* ?? *) and were subjected to second round analysis. The light chain variable region of antibody DS6 (Figure 13A) heavy chain variable region (Figure 13B). Figure 14 shows the map of mammalian plasmid prDS6 vl-0. This plasmid was used to construct and express recombinant and humanized chimeric DS6 antibodies. Figure 15 shows amino acid sequences of light chain (Figure 15A) and heavy chain variable domains (Figure 15A). 15B) of murine ("muDS0") and humanized ("huDS6") DS6 antibody (1.01 & 1.21). Figure 16 shows the cDNA and amino acid sequences of the light chain variable region for the humanized DS6 antibody ("huDS6") (1.01 and 1.21). Figure 17 shows the cDNA and amino acid sequences of the heavy chain variable region for the humanized DS6 antibody ("huDS6") 1.01 (Figure 17A) and 1.21 (Figure 17B). Figure 18 shows the flow cytometry binding curves of murine DS6 (muDSó) chimeric DS6 (chDS6), and human DS6 version 1.01 (huDS6 vl.01) and huDS6 version 1.21 (huDS6 vl.21) from a test executed in KB cells. The additives of murine, chimeric, and human antibodies vl.01 and vl.21 DS6 (muDSó = 0.82 nM, chDS6 = 0.69 nM, huDSóvl.01 -0.82 nM and huDS6vl.21 = 0.85 nM) are comparable, indicating that the coating has not decreased avidity. Figure 19 shows the results of a competitive binding test of muDS6, chDS6, huDS6 vl.01 and huDS6 vl .21 antibodies with biotinylated muDS6. The variable concentrations of muDSó, chDS6, huDSóvl.01 and huDSßv 1.21 discovered were combined with 2 nM of biotin-muDS6 and streptavidin-secondary DTAF. The IC50's (muDS6 = 1.9 nM, chDSó = 1.7 nM, huDSovl.01 = 3.0 nM, and huDS6vl.21 = 1.9 nM) of all antibodies are similar, indicating that humanization has not reduced avidity.

Figure 20 shows the results of a binding affinity determination of unconjugated DS6 antibody against a DS6-DMI antibody conjugate. The results demonstrated that DMI conjugation does not adversely affect the binding affinity of the antibody. The apparent Kd of the DS6-DMI antibody conjugate (3,902 nM) ("DS6-DM1") was slightly larger than the uncoated antibody (2020 nM) ("DS6"). Figure 21 shows the results of an indirect cell viability test using the DS6 antibody in the presence or absence of the conjugate anti-mouse IgG (H + L) DMI (2 ° Ab-DMI). Positive Caov-3 antigen cells were removed in a manner dependent on the DS6 antibody (IC50 = 424.9 pM) only in the presence of the secondary conjugate ("DS6 + 2 ° Ab-DMI"). Figure 22 shows the results of a complement dependent cytotoxicity (CDC) test of the muDS6 antibody. The results demonstrate that there is no CDC effect mediated by the DS6 antibody or in HPAC cells (Figure 22A) and ZR-75-1 (Figure 22B). Figure 23 shows the results of an in vitro cytotoxicity test of a DS6-DMI antibody conjugate against free maytansine. In a clonogenic test, the DS6 antigen-positive (Figure 23A), breast (Figure 23B), cervical (Figure 23C), and pancreatic (Figure 23D) ovarian cancer cell lines were tested for continuous exposure cytotoxicity for a conjugate of antibody DS6-DMI (left panels). These lines Cells were tested similarly for maytansine sensitivity by means of a 72h exposure for free maytansine (right panels). The 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-II. Figure 24 shows the results of an in vitro cytotoxicity test for a DS6-DMI antibody conjugate. In a MTT cell viability test, human ovarian cancer cells (Figure 24A, Figure 24B &Figure 24C), breast (Figure 24D &Figure 24E), cervical (Figure 24F &Figure 24G), and pancreatic (Figure 24H &Figure 241) were eliminated in a manner dependent on the DS6-DMI antibody conjugate. The uncoated DS6 did not adversely affect the growth of these cells, which indicate that the DMI conjugate is required for cytotoxicity. Figure 25A shows the results of an in vivo anti-tumor efficacy study of a DS6-DMI antibody conjugate in established subcutaneous KB tumor xenografts. The tumor cells were inoculated on day 0, and the first treatment was given on day 6. The immunoconjugate treatments continued daily for a total of 5 doses. The PBS control animals were sacrificed once the tumor volumes exceeded 1500 mm3. The conjugate was supplied in a dose of 150 or 225 μg / kg DMI, which corresponds to the antibody concentrations of 5.7 and 8.5 mg / kg respectively. The body weights (Figure 25B) of the mice were monitored during the course of the study. Figure 26 shows the results of an anti-tumor efficacy study of a DS6-DMI antibody conjugate in established subcutaneous tumor xenografts. OVCAR5 cells (Figure 26A and Figure 26B), TOV-21G (Figure 26C and Figure 26D), HPAC (Figure 26E and Figure 26F), and HeLa (Figure 26G and Figure 26H) were inoculated on day 0, and the treatments of immunoconjugate were supplied on day 6 and 13. The PBS control animals were sacrificed once the tumor volumes exceeded 1000 mm3. The conjugate was delivered at a dose of 600 μg / kg DMI, which corresponds to an antibody concentration of 27.7 mg / kg. Tumor volume (Figure 26A, Figure 26C, Figure 26E, and Figure 26G) and body weight (Figure 26B, Figure 26D, Figure 26F, and Figure 26H) of the mice were monitored during the course of the study. Figure 27 shows the results of an in vivo efficacy study of a muDS6-DMI antibody conjugate in intraperitoneal OVCAR5 tumors. The tumor cells were injected intraperitoneally on day 0, and the immunoconjugate treatments were given on day 6 and 13. The animals were sacrificed once the body weight exceeded 20%. Figure 28 shows the cytometry binding curve from of a binding affinity study of uncoated DS5 antibody and taxane-conjugated on HeLa cells. The taxane conjugation (MMI-202) does not adversely affect the binding affinity of the antibody. The apparent Kd of the conjugate DS6-MM1-202 (1.24 nM) was slightly larger than the uncoated DS6 antibody (620 pM).

Figure 29 shows the binding and the in vitro potency of the humanized DS6 antibody conjugate DS6 version 1.01. the conjugation of huDSóvl.01 with DM4 has little effect on the avidity of huDSóvl.01 of KB cells (Figure 29A). huDS6vl.01 -DM4 shows a potent in vitro cytotoxicity towards WISH cells that express DS6 with an IC50 of 0.44 nM (Figure 29B). Figure 30 shows the results of an in vivo efficacy study with conjugate huDSßvl.01 -DM4 in a pancreatic cancer model HPAC. huDSóvl .01 -DM4 showed potent antitumor activity whereas the control conjugate B4-DM4 whose target is not expressed in the HPAC model essentially did not have activity (Figure 30A). The administered dose of 200 μg / kg was not toxic for the animals as indicated by the lack of weight loss (Figure 30B).DETAILED DESCRIPTION OF THE INVENTION The present invention provides other features, monoclonal anti-CA6 antibodies, humanized anti-CA6 antibodies, and fragments of anti-CA6 antibodies.

Each of the antibodies and antibody fragments of the present invention are designed to specifically recognize and bind the glycoprote CA6 on the surface of a cell. It is known that CA6 is expressed by many human tumors: 95% of serous ovarian carcinomas, 50% of endometrioid ovarian carcinomas, 50% of uterine cervix neoplasms, 69% of endometrial neoplasms, 80% of neoplasms of the vulva, 60% of breast carcinomas, 67% of pancreatic tumors, and 48% of urothelial tumors, although it is rarely expressed by normal human tissue. A report by Kearse et al., Int. J. Cancer 88 (6): 866-872 (2000) erroneously identified the protein on which the CA6 epitope was found as an 80 kDa protein having an N-linked carbohydrate containing the CA6 epitope when they are used using a hybridoma supernatant to characterize it. Using purified DS6 it has been shown that the CA6 epitope is found in an O-linked carbohydrate of more than 250 kDa non-disulfide linked glycoprotein. In addition, the glycoprotein was identified as mucin, Mucl. Because different Mucl alleles have varying numbers of tandem repeats in the variable number tandem repeat domain (VNTR) cells they frequently express two different Mucl proteins of different sizes (Taylor-Papadimitriou, Biochim, Biophys, Acta 1455 (2 -3): 301 -13 (1999) .Due to the differences in the number of repetitions in the VNTR domain as well as the differences in glycosylation the molecular weight of Mucl varies from one cell line to another. The susceptibility of CA6 immunoreactivity to periodic acid indicates that CA6 is a "carbohydrate epitope glycopetope". The additional susceptibility of CA6 immunoreactivity to treatment with neuraminidase from Vibrio cholerae indicates that the CA6 epitope is a glycoprotein dependent on sialic acid, therefore it is a "sialoglicatope". The details of the characterization of CA6 can be found in Example 2 (see below). Additional details on CA6 can be found in WO 02/16401; Wennerberg et al., Am. J. Pathol. 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. J. Gynecol Pathol. 20 (3): 260-6 (2001); and Smith et al., Appl. Immunohistochem. Mol. Morphol. 10 (2): 152-8 (2002). The present invention also includes cytotoxic conjugates comprising two primary components. The first component is a cell binding agent that recognizes and binds the CA6 glycopept. The cell binding agent will recognize the CA6 sialogliquetope in Muc 1 with a high degree of specificity so that the cytotoxic conjugates recognize and bind only the cells for which they are intended. A high degree of specificity will allow the conjugates to act in a targeted manner with few side effects that result from the non-specific binding. In another embodiment, the cell binding agent of the present invention also recognizes the CA6 glycopeto with a high degree of affinity so that the conjugates will be in contact with the target cell for a time sufficient to allow the cytotoxic drug portion of the conjugate to act on the cell, and / or to allow to the conjugates sufficient time in which they are internalized by the cell. In a preferred embodiment, the cytotoxic conjugates comprise anti-CA6 antibody as the cell binding agent, most preferably the murine anti-CA6 monoclonal antibody DS6. In a more preferred embodiment, the cytotoxic conjugates comprise a humanized DS6 antibody or an epitope-binding fragment thereof. The DS6 antibody is able to recognize CA6 with a high degree of specificity and directs the cytotoxic agent towards an abnormal cell or tissue, such as cancer cells, in a targeted manner. The second component of the cytotoxic conjugates of the present invention is a cytotoxic agent. In preferred embodiments, the cytotoxic agent is a taxol, a maitansionoid such as DMI or DM4, CC-1065 or a CC-1065 analog. In preferred embodiments, the cell binding agents of the present invention are covalently linked, directly or through a separable or non-separable linker, to the cytotoxic agent. Cell binding agents, cytotoxic agents, and linkers are described in greater detail below.

Cell Link Aqentes The effectiveness of the compounds of the present invention as therapeutic agents depends on the careful selection of an appropriate cell binding agent. The cell binding agents may be of any class currently known, or to be known, and include peptides and non-peptides. The cell binding agent can be any compound that can bind a cell, either in a specific or non-specific manner. In general, these can be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (such as transferin), or any other molecule or cell-binding substance.

More specific examples of cell binding agents that can be used include: (a) polyclonal antibodies; (b) monoclonal antibodies; (c) antibody fragments such as Fab, Fab ', and F (ab') 2, Fv (Parham, J. Immunol., 131: 2895-2902 (1983); Spring et al., J. Immunol., 113: 470- 478 (1974); Nisonoff et al., Biochem Biophys., 89: 230-244 (1960)); (d) interferons (e.g., alpha, beta, .gamma.); (e) lymphokines such as IL-2, IL-3, IL-4, IL-6; (f) hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte stimulation hormone), steroid hormones, such as androgens and estrogens; (g) growth factors and colony stimulation factors such as EGF, TGF-alpha, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984)); (h) transferin (O'Keefe et al., J. Biol. Chem. 260: 932-937 (1985)); and (i) vitamins, such as folate.

Antibodies The selection of the appropriate cell binding agent is a matter of choice that depends on the particular cell population to which it is directed, although in general, antibodies are preferred if available or an appropriate one, more preferably an antibody, can be prepared monoclonal The monoclonal antibody techniques allow the production of extremely specific cell binding agents in the form of specific monoclonal antibodies. The techniques for creating monoclonal antibodies produced through the immunization of mice, rats, guinea pigs or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, viruses are particularly well known. complete, attenuated complete viruses and viral proteins such as viral coat proteins. Human sensitized cells can also be used. Another method for creating monoclonal antibodies is the use of phage libraries of scFv (individual chain variable region), specifically human scFv (see, for example, Griffiths et al., U.S. Patent Nos. 5,885,793 and 5,969,108; McCafferty et al. ., WO 92/01047; Liming et al., WO 99/06587). A typical antibody is comprised of two identical heavy chains and two identical light chains that are linked by disulfide bonds. The variable region is a portion of the heavy chains and light chains of the antibody that differs in sequence between the antibodies and that cooperates in the binding and specificity of each particular antibody to its antigen. Usually, the variability is not evenly distributed across the variable regions of the antibody. It is commonly concentrated within three segments of a variable region called complementation determining regions (CDRs) or hypervariable regions, both in the light chain and heavy chain variable regions. The most highly conserved portions of the variable regions are called structure regions. The variable regions of heavy and light chains comprise four framework regions, which largely adopt a lamin-beta configuration, with each region of structure connected by the three CDRs, which form a cycle connecting the beta-sheet structure and , in some cases, they are part of the beta-sheet structure. The CDRs in each chain are held in close proximity by the structure regions and, with the CDRs of the other chain, they contribute to the formation of the antigen binding site of the antibodies (E. A. Kabat et al Sequences of Proteins of Immunological Interest, Fifth Edition, 1991, NIH). The constant region is a portion of the heavy chain. Insofar as it is not directly involved in the binding of an antibody to an antigen, it exhibits several effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity. A monoclonal antibody suitable for use in the present invention includes the murine monoclonal antibody DS6 (U.S. Patent No. 6,596,503, ATCC Deposit Number PTA-4449).

Humanized or Coated DS6 Antibodies Preferably, a humanized anti-CA6 antibody is used as the cell binding agent of the present invention. A preferred embodiment of said humanized antibody is a humanized DS6 antibody, or an epitope binding fragment thereof. The goal is a reduction in the immunogenicity of a xenogeneic antibody, such as a murine antibody, for introduction into a human, while maintaining complete antigen binding affinity and the specificity of the antibody.

Humanized antibodies can be produced using various technologies such as coating and CDR grafting. As used herein, the coating technology employs a combination of molecular modeling, statistical analysis and mutagenesis to alter the non-CDR surfaces of the antibody variable regions so that they resemble the surfaces of known antibodies of the target receptor. Strategies and methods for antibody coating, and other methods for reducing the immunogenicity of antibodies within a different receptor, are described in U.S. Patent 5,639,641 (Pedersen et al.), Which is incorporated herein by reference. the present by reference in its entirety. Briefly, in a preferred method, (1) the positional alignments of a deposition of heavy and light chain variable regions of antibody are generated to give a set of exposed positions of structure surface and heavy and light chain region where the positions of alignment for all variable regions are at least approximately 98% identical; (2) a set of exposed amino acid residues of heavy and light chain variable region structural surface is defined by a rodent antibody (or fragment thereof); (3) a set of exposed amino acid residues of heavy and light chain variable region structural surface is identified that is closely similar to the set of exposed amino acid residues of rodent surface; (4) the The set of exposed amino acid residues of heavy and light chain variable region structural surface defined in step (2) is substituted with the set of exposed amino acid residues of heavy and light chain variable region structural surface identified in step ( 3), except for those amino acid residues that are within 5 A of any atom of any residue of the complementation determining regions of the rodent antibody; and (5) the humanized rodent antibody having binding specificity is produced.

The antibodies can be humanized using a variety of other techniques including CDR-grafting (EP 0 239 400; WO 91/09967; US Patents Nos. 5,530,101; and 5,585,089), coating or coating (EP 0 592 106; EP 0 519 596; Padlan EA, 1991, Molecular Immunology 28 (4/5): 489-498; Studnicka GM et al., 1994, Protein Engineering 7 (6): 805-814; Roguska MA et al., 1994 , PNAS 91: 969-973), and chain redistribution (US Pat. No. 5,565,332). Human antibodies can be produced from a variety of methods known in the art including phage display methods. See also Patents of the United States of North America Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patent application publications numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said references incorporated in their totality by reference).

In a preferred embodiment, the present invention provides humanized antibodies or fragments thereof that recognize a novel sialoglicatope (the CA6 glycopetope) in Mucl mucin. In another embodiment, the humanized antibodies or epitope-binding fragments thereof have the additional ability to inhibit the growth of a cell expressing the CA6 glycopept. In more preferred embodiments, coated or humanized versions of the DS6 antibody are provided wherein the surface-exposed residues of the antibody or its fragments are replaced in both light and heavy chains to more closely resemble known human antibody surfaces. Humanized DS6 antibodies or epitope binding fragments thereof of the present invention have improved properties. For example, humanized DS6 antibodies or epitope-binding fragments thereof specifically recognize a novel sialoglicatope (the CA6 glycopetope) in Mucl mucin. Most preferably, the humanized DS6 antibodies or epitope-binding fragments thereof have the additional ability to inhibit the growth of a cell expressing the CA6 glycopod. The humanized antibody or an epitope binding fragment thereof can be conjugated to a drug, such as a maitansionoid, to form a prodrug having specific cytotoxicity towards the antigen-expressing cells by targeting the novel Mucl sialogliquetope, CA6. The cytotoxic conjugates comprising said antibodies and some highly toxic drug (eg, maytansinoids, taxanes, and CC-1065 analogues) can be used as a therapeutic for the treatment of tumors, such as breast and ovarian tumors. The humanized versions of the DS6 antibody are also fully characterized herein with respect to their respective amino acid sequences of both light and heavy chain variable regions, the DNA sequences of the genes for the light and heavy chain variable regions, the identification of the CDRs, the identification of their surface amino acids, and the description of a means for their expression in recombinant form. In one embodiment, a humanized antibody or epitope binding fragment thereof having a heavy chain including CDRs having amino acid sequences represented by NOS SEQ ID: 1-3: SYNMH (NO ID SEC: 1) YIYPGNGATNYNQKFKG ( NO SEC ID: 2) GDSVPFAY (NO SEC ID: 3) When the heavy chain CDRs are determined by the AbM modeling software they are represented by SEQ ID NOS: 20-22: GYTFTSYNMH (NO SEC ID: 20) YIYPGNGATN (NO SEC ID: 21) GDSVPFAY (NO SEC ID: 22) In the same embodiment, the humanized antibody or fragment of epitope binding thereof has a light chain comprising CDRs having amino acid sequences represented by NOS SEC ID: 4-6: SAHSSVSFMH (NO SEC ID: 4) STSSLAS (NO SEC ID: 5) QQRSSFPLT (NO SEC ID: 6) Humanized antibodies and epitope-binding fragments thereof having a light chain variable region having an amino acid sequence that shares at least 90% sequence identity with an amino acid sequence represented by NO ID are also provided. SEC: 7 or NO SEC ID: 8: QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLW IYSTSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQSSFPLT FG AGTKLELKR. (NO SEC ID: 7) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKL WIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPL TFG AGTKLELKR. (SEQ ID NO: 8) Similarly, humanized antibodies and epitope-binding fragments thereof are provided which have a heavy chain variable region having an amino acid sequence that shares at least 90% sequence identity with an amino acid sequence represented by NO SEC ID: 9, NO SEC ID: 10, or NO SEC ID: 11: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEWIGYIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO: 9) QAQLVQSGAEWKPGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVY FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYF CARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO: 11) In another embodiment, humanized antibodies and epitope-binding fragments thereof are provided which have a humanized or coated light chain variable region possessing an amino acid sequence corresponding to NO SEQ ID: 8 EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKL WIYSSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLT FG AGTKLELKR. (SEQ ID NO: 8) Similarly, humanized antibodies and epitope-binding fragments thereof are provided which have a humanized or coated heavy chain variable region possessing an amino acid sequence corresponding to NO SEQ ID NO: 10 or NO SEC ID: 11: QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEIG YGYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCAR GDSVPFAYWGQGTLVTVSA. (NO SEC ID: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCAR GDSVPFAYWGQGTLVTVSA. (SEQ ID NO: 11) Humanized antibodies and epitope-binding fragments thereof of the present invention may also include versions of light and / or heavy chain variable regions in which the amino acid residues of human surface in proximity the CDRs are replaced by the corresponding muDS6 surface residues in one or more positions defined by the residues in Table 1 (Kabat numbering) marked with an asterisk in order to retain the binding affinity and the specificity of muDSó. Table 1 The primary amino acid and DNA sequences of the heavy and light chains of the DS6 antibody, and of numanized versions thereof, are described herein. However, the scope of the present invention is not limited to antibodies and fragments who understand these sequences. Instead, all antibodies and fragments that specifically bind to CA6 as the only tumor-specific glycogen in the Mud receptor are included in the present invention. Preferably, antibodies and fragments that specifically bind to CA6 also antagonize the biological activity of the receptor. More preferably, said antibodies substantially also lack antagonist activity. Therefore, the antibodies and antibody fragments of the present invention may differ from the DS6 antibody or humanized derivatives thereof, in the amino acid sequences of their support, CDRs, and / or light chain and heavy chain and still remain within of the scope of the present invention. The CDRs of the DS6 antibody are identified by means of modeling and their molecular structures have been predicted. Again, while the CDRs are important for epitope recognition, they are not essential for the antibodies and fragments of the invention. Accordingly, antibodies and fragments having improved properties produced by, for example, affinity maturation of an antibody of the present invention are provided. The light chain of mouse Ig VK ap4 germline gene and the heavy chain IgVh J558.41 germline gene from which DS6 was probably derived are shown in FIGURE 11 aligned with the DS6 antibody sequence. The comparison identifies probable somatic mutations in the DS6 antibody, including several in the CDRs. The sequence of the light chain and heavy chain variable region of the DS6 antibody, and the sequences of the CDRs of the DS6 antibody was not known previously and is set forth in Figures 9A and 9B. This information can be used to produce humanized versions of the DS6 antibody.

Antibody Frames The antibodies of the present invention include the full-length antibodies described above, as well as epitope-binding fragments. As used herein, "antibody fragments" include any portion of an antibody that retains the ability to bind to the epitope recognized by the full-length antibody, generally referred to as "epitope-binding fragments." Examples of antibody fragments include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (dsFv) and fragments that they comprise a region VL or VH region. The epitope binding fragments, which include individual chain antibodies, may comprise the variable region (s) alone or in combination with all or a portion of the following: joint region, CHI , CH2, and CR3 domains. These fragments may contain one or both fragments Fab or fragment F (ab ') 2. Preferably, the antibody fragments contain the six CDRs of the complete antibody, although the fragments containing less of those regions, such as the three, four or five CDRs, are also functional. In addition, the fragments may be or may combine members of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof. The Fab and F (ab ') 2 fragments can be produced through proteolytic sequencing, utilizing enzymes such as papain (Fab fragments) or pepsin fragments (F (ab') 2). Single chain FVs (scFvs) fragments are epitope binding fragments containing at least one fragment of an antibody heavy chain variable region (VH) linked to at least one fragment of an antibody light chain variable region. (VL) The linker can be a short, flexible peptide selected to ensure that adequate three-dimensional folding of the (V) and (VH) regions occurs once they are linked in order to maintain the target-to-target molecule-specificity of the entire antibody from which the single chain antibody fragment was derived. The carboxyl terminus of the sequence (VL) or (VH) can be covalently linked to the amino acid term of a complementary sequence (VL) or (VH).

The single chain antibody fragments of the present invention contain amino acid sequences having at least one of the variable or determinant regions of complementation (CDRs) of the complete antibodies described in this specification, although they lack some or all of the constant domains of those antibodies. These constant domains are not necessary for the antigen binding, although they constitute a major portion of the structure of the complete antibodies. The single chain antibody fragments can therefore supercharge some of the problems associated with the use of antibodies that contain a part or all of the constant domain. For example, single chain antibody fragments tend to be free of undesirable interactions between biological molecules and the heavy chain constant region, or other undesirable biological activity. Additionally, single chain antibody fragments are considerably smaller than whole antibodies and therefore may have higher capillary permeability than whole antibodies, allowing single chain antibody fragments to locate and bind to antigen binding sites. objective more efficiently. Also, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thereby facilitating their production. In addition, the relatively small size or fragments of single chain antibody are less likely to elicit an immune response in a recipient than whole antibodies. The individual chain antibody fragments can be generated through molecular cloning, the library of antibody phage display or similar techniques well known to the experienced technician. These proteins can be produced, for example, in eukaryotic cells or prokaryotic cells, including bacteria. The epitope binding fragments of the present invention can also be generated using various methods of phage display known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. In particular, said phage can be used to display epitope binding domains expressed from a repertoire or combination antibody library (eg, human or murine). The phage expressing an epitope binding domain that binds the antigen of interest can be selected or identified with antigen, for example, using labeled or bound tagged antigen for a surface or solid bed. The phage used in these methods is commonly a filamentous phage that includes fd and Ml 3 binding domains expressed from phage with Fab, Fv or recombinantly fused disulfide-stabilized Fv domains fused to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the epitope binding fragments of the present 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; Kettleborough 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: 191-280; PCT application No. PCT / GB91 / 01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and Patents of the United States of North America Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety. After phage selection, the regions of the phage encoding the fragments can be isolated used to generate the epitope-binding fragments through expression in a selected receptor, including mammalian cells, insect cells, plant cells, yeast , and bacteria, using recombinant DNA technology, for example, as described in detail below. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art such as those described in PCT publication 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 references that are incorporated by reference in their entirety. Examples of techniques that can be used to produce Fvs and single chain antibodies include those described in the Patents of the United States of North America 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 The functional equivalents of the anti-CA6 antibody and the humanized anti-CA6 antibody are also included within the scope of the invention. The term "functional equivalents" includes antibodies with homologous sequences, chimeric antibodies, artificial antibodies and modified antibodies, for example, wherein each functional equivalent is defined by its ability to bind to CA6. The experienced technician will understand that there is an overlap in the group of molecules called "antibody fragments" and the group called "functional equivalents". Methods for producing functional equivalents are described, for example in, for example, PCT Application WO 93/21319, European Patent Application No. 239,400; PCT Application WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424, which are incorporated in their entirety by reference. Antibodies with homologous sequences are those antibodies with amino acid sequences that have sequence homology with the amino acid sequence of an anti-CA6 antibody and a humanized anti-CA6 antibody of the present invention. The preferable homology is with the amino acid sequence of the variable regions of the anti-CA6 antibody and humanized anti-CA6 antibody of the present invention. The "sequence homology" as applied to an amino acid sequence herein is defined as a sequence with at least about 90%, 91%, 92%, 93%, or 94% sequence homology, and more preferably of at least about 95%, 96%, 97%, 98%, or 99% sequence homology for another amino acid sequence, as determined, for example, by the FASTA search method according to Pearson and Lipman, Proc. Nati Acad. Sci. USA 85, 2444-2448 (1988). As used herein, a chimeric antibody is one in which different portions of an antibody are derived from different animal species. For example, an antibody having a variable region derived from a murine monoclonal antibody paired with a constant region of human immunoglobulin. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; Patents of the United States of North America Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized forms of chimeric antibodies are made through the replacement of the complementation determining regions of, for example, a mouse antibody, in a human structure domain, for example, see PCT Publication No. W092 / 22653. The humanized chimeric antibodies preferably have constant regions and variable regions different from the complementation determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and the CDRs derived substantially or exclusively from a mammal other than the human human. Artificial antibodies include scFv fragments, diabodies, triabodies, tetrabodies and mru (see reviews by Winter, G. and Milstein, C, 1991, Nature 349: 293-299, Hudson, PJ., 1999, Current Opinion in Immunology 11: 548 -557), each of which has the ability to bind antigen. In the single chain Fv fragment (scFv), the VH and VJ domains of an antibody are linked through a flexible peptide. Commonly, this linker peptide has a length of about 15 amino acid residues. If the linker is much smaller, for example 5 amino acids, diabodies are formed, which are bivalent scFv dimers. If the linker is reduced to less than three amino acid residues, trimeric and tetrameric structures are formed which are called triabodies and tetrabodies. The smallest binding unit of an antibody is a CDR, commonly the heavy chain CDR2 that has sufficient recognition and specific binding that can be used by separated. This fragment is called the molecular recognition unit or mru. Several mrus can be linked together with short linker peptides, thus forming a binding protein artificial linker with greater avidity than an individual mru. The functional equivalents of the present application also include modified antibodies, for example, antibodies modified by the covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic separation, binding to cellular ligand or other protein, etc. . Covalent binding does not prevent the antibody from generating an anti-idiotypic response. These modifications can be carried out through known techniques, including, but not limited to, specific chemical separation, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the modified antibodies may contain one or more non-classical amino acids. Functional equivalents can be produced by exchanging different CDRs in different chains within different structures. Therefore, as an example, different classes of antibody are possible for a given set of CDRs through the substitution of different heavy chains, whereby, for example, types and isotypes of antibody IgGI-4, IgM, can be produced, IgA-2, IgD, IgE. Similarly, antibodies artificial within the scope of the invention can be produced by incrustation of a set of CDRs within a completely synthetic structure. Functional equivalents can be easily produced through mutation, deletion and / or insertion within variable and / or constant region sequences flanking a particular set of CDRs, using a wide variety of methods known in the art. The antibody fragments and functional equivalents of the present invention encompass those molecules with a detectable degree of binding to CA6, when compared to the DS6 antibody. A detectable degree of linkage includes all values in the range of at least 10-100%, preferably at least 50%, 60% or 70%, most preferably at least 75%, 80%, 85%, 90%, 95% or 99% of the binding capacity of the murine DS6 antibody to CA6.

Improved Antibodies CDRs are of primary importance for epitope recognition and antibody binding. However, changes can be made to the residues comprising the CDRs without interfering with the ability of the antibody to recognize and bind its cognate epitope. For example, changes that do not affect the recognition of the epitope can be made, increase the affinity of antibody binding for the epitope. Therefore, also included in the scope of the present invention are the improved versions of both murion and humanized antibodies, which recognize and bind in the same way and specifically CA6, preferably with increased affinity.

Several studies have compiled the effects of the introduction of one or more amino acid changes in several positions in the sequence of an antibody, based on the knowledge of the primary antibody sequence, in its properties such as link and level of expression (Yang , WP et al., 1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc. Nati, Acad. Sci. USA, 95, 8910-8915; Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535-539). In these studies, the primary antibody equivalents have been generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2, CDR3, or framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, mutagenesis of cartridge, PCR prone to error, DNA castellation, or mutator-strains of E. coli (Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535- 539; Adey, NB et al., 1996, Chapter 16 , pp. 277-291, in "Phage Display of Peptides and Proteins", Eds. Kay, BK et al., Academic Press). These methods for changing the primary antibody sequence have resulted in improved affinities of the secondary antibodies (Gram, H. et al., 1992, Proc Nati Acad Sci USA, 89, 3576-3580, Boder, ET et al. ., 2000, Proc. Nati, Acad. Sci. USA, 97, 10701- 10705; Davies, J. and Riechmann, L., 1996, Immunotechnolgy, 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. Biol. Chem., 276, 27622-27628). By means of a similar directed strategy of changing one or more amino acid residues of the antibody, the antibody sequences described in this invention can be used to develop the anti-CA6s antibody with improved functions, including improved affinity for CA6. Enhanced antibodies also include those antibodies that have improved characteristics than with preparations through standard animal immunization techniques, hybridoma formation and selection of antibodies with specific characteristics.

Cytotoxic acids The cytotoxic agent used in the cytotoxic conjugate of the present invention can be any compound that results in the death of a cell, or induces cell death, or in some way decreases cell viability. Preferred cytotoxic agents include, for example, maytansinoids and maytansinoid analogues, taxoids, CC-1065 and analogues CC-1065, dolastatin and dolastatin analogues, defined below. These cytotoxic agents are conjugated to antibodies, antibody fragments, functional equivalents, improved antibodies and their analogues.

Cornos and describes in the present. The cytotoxic conjugates can be prepared through in vitro methods. In order to bind a drug or a prodrug to the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, labile acid groups, forlabile groups, labile peptidase groups and labile esterase groups. Preferred linking groups are disulfide groups and thioether groups. For example, the conjugates can be made using a disulfide exchange reaction or through the formation of a thioether bond between the antibody and the drug or prodrug.

Mayansinosides Among the cytotoxic agents that can be used in the present invention to form a cytotoxic conjugate are the maytansinoids and maytansinoid analogues. Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells. Examples of suitable maytansinol analogs include those having a modified aromatic ring and those having modifications in other positions. Suitable maytansinoids are described in U.S. Pat.

North America Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545. Specific examples of suitable maytansinol analogues having aromatic ring include: (1) C-19-dechloro (U.S. Patent No. 4,256,746) (prepared by LAH reduction of ansamitocin P2); (2) C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH ); and (3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/- decloro (U.S. Patent No. 4,294,757) (prepared by acylation using acyl chloride). Specific examples of maytansinol analogs having modifications of other positions include: (1) C-9-SH (US Pat. No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5); (2) C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Patent No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Patent No. 4,450,254) (prepared by Nocardia); (4) C-15-hydroxy / acyloxy (U.S. Patent No. 4,364,866) (prepared by the conversion of maytansinol by means of Streptomyces); (5) C-15-methoxy (Patents of the United States of North America Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora); (6) C-18-7V-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol through Streptomyces); and (7) 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol). In a preferred embodiment, the cytotoxic conjugates of the present invention utilize the thiol-containing maitansionoid (DMI), formerly designated N 2 -deacetyl-N 2 '- (3-mercapto-1-oxopropyl) -maitansine, as the cytotoxic agent. DMI is represented by the following structural formula (I): In another preferred embodiment, the cytotoxic conjugates of the present invention utilize the thiol-containing maytansinoid N2-deacetyl-N- (4-methyl-4-mercapto-1-oxopentyl) -maitansin as the cytotoxic agent. DM4 is represented by the following structural formula (II): In further embodiments of the invention, other maytansins, including thiol and maytansinoids containing disulfide having a mono- or di-alkyl substitution at the carbon atom held by the sulfur atom may be used. These include a maitansionoid that has C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, an acylated amino acid side chain with an acyl group having a hindered sulfhydryl group, wherein the carbon atom of the acyl group having the thiol functionality possesses one or two substituents, substituents which are CH3, C2H5, straight or branched alkyl or alkenyl having from 1 to 10 carbon atoms, alkyl or cyclic alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or radical heterocyclic or aromatic heterocyclic, and wherein in addition one of the substituents may be H, and wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl functionality and the sulfur atom. Said additional maytansines including compounds represented by the formula (III): where: Y 'represents (CR7CR8)? (CR9 = CR1o) pC = CqAr (CR5CR6) mDu (CR11-CR? 2) r (C = C) sBt (CR3CR4) nCR? R2 sz, where: Ri and R2 are each independently CH3, C2Hs, linear 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 radical heterocyclic aromatic or heterocycloalkyl, and further R2 can be H; A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl or heterocyclic aromatic radical or heterocycloalkyl; R3 > R4 > Rs, Re-R7, R8, Rc, R1, and R12 are each independently H, CH3, C2H5, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl which have from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic radical or heterocycloalkyl; I, 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 1, m, n, o, p , q, r, syt are not zero at any time; and Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic radical or heterocycloalkyl. Preferred embodiments of the formula (III) include compounds of the formula (III) wherein: Ri is H, R2 is methyl and Z is H. Ri and R2 are methyl and Z is H. Ri is H, R2 is methyl, and Z is -SCH3. Ri and R2 are methyl, and Z is -SCH3. Said additional maytansins also include compounds represented by the formulas (IV-L), (IV-D), or (IV-D.L): wherein: Y represents (CR7CRβ) i (CR5CR6) m (CR3CR4) nCR1R2SZ, wherein: R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from 1 to 10 carbon atoms, alkyl or branched or cyclic alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, and further R2 can be H; R3, R4, R5, Re. R7 and R8 are each independently H, CH3, C2H5, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 atoms carbon, phenyl, substituted phenyl, or heterocyclic aromatic radical or heterocycloalkyl; I, m and n are each independently an integer from 1 to 5, and in addition n can be 0; Z is H, SR or -COR wherein R is linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, alkyl or cyclic alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic radical or heterocycloalkyl; and May represents a maitansionoid that has the side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl. Preferred embodiments of the formulas (IV-L), (IV-D) and (IV-D5L) include compounds of the formulas (FV-L), (FV-D) and (FV-D, L) where: is H, R2 is methyl, R5, R6, R7, and Rβ each is H, each of I and m are 1, n is 0, and Z is H. Ri and R2 are methyl, R5, R6, R7, R8 are each H, I and m are 1, n is 0, and Z is H. Ri is H, R2 is methyl, R5, R6, R7, and R8 each is H, each of I and m is 1, n is 0, and Z is -SCH3. Ri and R2 are methyl, R5, R6, R7, R8 each is H, I and m are 1, n is 0, and Z is -SCH3. Preferably, the cytotoxic agent is represented by the formula (FV-L). Said additional maytansins also include compounds represented by the formula (V): (V) where: Y represents (CR7CR8) i (CR5CR6) p, (CR3CR4) r, CRiR2SZ, where: Ri and R2 are each independently CH3, C2H5, linear 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 aromatic radical heterocyclic or heterocycloalkyl, and further R2 may be H; R3, R4. Rs, Rß > R7 and R8 are each independently H, CH3, C2H5, linear 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 radical or heterocycloalkyl; I, m and n are each independently an integer from 1 to 5, and in addition n can be 0; and Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic radical or heterocycloalkyl. Preferred modaldiades of the formula (V) include compounds of the formula (V) wherein: R1 is H, R2 is methyl, R5, R6, R7, and R8 each is H; each of I and m is 1; n is 0; and Z is H. R1 and R2 are methyl; R5, R6, R7, R8 each is H, I and m are 1; n is 0; and Z is H. R1 is H, R2 is methyl, R5, R6, R7, and R8 each is H, each of I and m is 1, n is 0, and Z is -SCH3. RI and R2 are methyl, R5, R6, R7, R8 each is H, I and m are 1, n is 0, and Z is -SCH3. Said additional maytansins further include compounds represented by the formula (Vl-L), (Vl-D), or (VI-D.L): (Vl-L) (VI-D) (VI-D,) wherein: Y2 represents (CR7CR8)? (CR5CRe) 171 (CR3CR4) HCR1R2SZ2, wherein: R and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from 1 to 10 carbon atoms, alkyl or branched or cyclic alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic radical or heterocycloalkyl, and further R2 can be H; R3, R4 > R5, Rd > R and Re are each independently H, CH3, C2H5, linear or cyclic alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, phenyl substituted or aromatic heterocyclic radical or heterocycloalkyl; I, m and n are each independently an integer from 1 to 5, and in addition n can be 0; Z2 is SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic radical or heterocycloalkyl; and May is a maitansionoid. Said additional maytansins also include compounds represented by the formula (VII): where: Y2 'represents (CR7CR8)? (CR9 = CR1O) P (C = C) qAr (CR5CR6) mDlJ (CR11 = CR12) r (C = C) and Bt (CR3CR4) nCR1R2S- R2SZ2, where: Ri and R2 are each independently CH3, C2H5, straight or branched alkyl or alkenyl having from 1 to 10 carbon atoms, alkyl or cyclic alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or aromatic radical heterocyclic or heterocycloalkyl, and further R2 may be H; A, B, and D each is independently cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic radical or heterocycloalkyl; R3. R > Rs, Re. R7 > Re. RT > R n, and R 2 are each independently H, CH 3, C 2 H 5, linear 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 radical or heterocycloalkyl; I, 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 I, m, n, o, p , q, r, syt are not zero at any time; and Z is SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3-10 carbon atoms, or simple or substituted aryl or aromatic radical heterocyclic or heterocycloalkyl. Preferred embodiments of the formula (VII) include compounds of the formula (VII) wherein: R1 is H and R2 is methyl. Maytansinoids previously mentioned may be conjugates for anti-CA6 DS6 antibody, or a homologue or fragment thereof, wherein the antibody is linked to maytansinoid using the thiol or disulfide functionality that is present in the acyl group of an acylated amino acid side chain found in C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl of maytansinoid, and wherein the acyl group of the laterald and acylated amino acid chain has its thiol or disulfide functionality located on a carbon atom having one or two substituents, said substituents which are CH3, C2H5, linear 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 aromatic heterocyclic radical or heterocycloalkyl, and furthermore one of the substituents may be H, and wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl functionality and the sulfur atom. A preferred conjugate of the present invention is one comprising the anti-CA6 DS6 anti-antibody, or a homologue or fragment thereof, conjugated to a maitansionoid of the formula (VIII): (vip) where: Y1 'represents (CR7CR8), (CR9 = CR10) p (C = C) qAr (CR5CR6) mDu (CR11 = CR12) r (C = C) s Bt (CR3CR4) nCR? R2S-, where: A, B, and D, each independently is cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic radical or heterocycloalkyl; R3. R > Rs, Re. R7, R, R9. Ru > and R 2 are each independently H, CH 3, C 2 H 5, linear 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 radical or heterocycloalkyl; and I, 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 I, m, n, o, p, q, r, syt are not zero at any time. Preferably, R is H and R 2 is methyl or R 1 and R 2 are methyl. An even more preferred conjugate of the present invention is one comprising the anti-CA6 DS6 antibody, or a homologue or fragment thereof, conjugated to a maitansionoid of the formula (IX-L), (IX-D), or (IX) -DL): where: Y1 represents (CR7CR8)? (CR5CR6) m (CR3CR4) nCRiR2S-, where: R? and R2 are each independently CH3, C2H5, linear 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, aromatic heterocyclic radical or heterocycloalkyl, and further R2 can be H; R3. R4 Rs. Rß > R7 and R1 are each independently H, CH3, C2H5, linear 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 0 heterocyclic aromatic radical or heterocycloalkyl; I, m and n are each independently an integer from 1 to 5, and also n can be 0; and May represents a maytansinol which has the side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl. Preferred embodiments of the formulas (IX-L), (IX-D) and (IX-D5L) include compounds of the formulas (DC-L), (DC-D) and (DC-D5L) where: R? is H and R2 is methyl or R? and R2 are methyl, R? is H, R 2 is methyl, R 5, R 6, R 7 and R 8 each is H; I and m each is 1; n is 0, R? and R2 are methyl; R5, R6, R7 and R8 each is H; I and m are 1; n is 0. Preferably, the cytotoxic agent is represented by the formula (IX-L). A further exemplified conjugate of the present invention is one comprising the anti-CA6 DS6 antibody, or a homologue or fragment thereof, conjugated to a maitansionoid of the formula (X): wherein the substituents are as defined for formula (IX) above. Any of the compounds described above are of special preference, where R? is H, R2 is methyl, R5, R6, R7 and R8 each is H, I and m each is 1, and n is 0. Especially additionally preferred are any of the compounds described above, wherein R and R2 are methyl, R5, R6, R7, R8 each is H, I and m are 1, and n is 0 In addition, the Z-aminoacyl steroisomer is preferred. Each of the maytansinoids shown in U.S. Patent Application No. 10 / 849,136, filed May 20, 2004, may also be used in the cytotoxic conjugate of the present invention. The complete description of the patent application of the United States of America number 10 / 849,136 is incorporated herein by reference.

Linking groups containing disulfide In order to bind the maytansinoid to a cell binding agent, such as the DS6 antibody, the maytansinoid comprises a linking portion. The binding portion contains a chemical bond that allows the release of the fully active maytansinoids at a particular site. Suitable chemical bonds are well known in the art and include disulfide bonds, labile acid bonds, photolabile bonds, labile peptidase bonds and labile esterase linkages. Disulfide bonds are preferred. The linking portion also comprises a reactive chemical group. In a preferred embodiment, the reactive chemical group can be covalently linked to the maytansinoid through a linking portion of the disulfide bond. Particularly preferred reactive chemical groups are N-succinimidyl esters and N-sulfosuccinimidyl esters.

Particularly preferred maytansinoids comprising a linking portion containing a reactive chemical group are C-3 maitansinol esters and their analogs wherein the linking portion contains a disulfide bond and the chemical reactive group comprises an N-succinimidyl ester or of N-sulfosuccinimidyl. Many positions in maytansinoids may serve as the position to chemically bind the binding portion. For example, the position C-3 having a hydroxyl group, the position C-14 modified with hydroxymethyl, the position C-15 modified with hydroxy and the position C-20 having a hydroxy group are expected to be useful. However, the C-3 position is preferred and the C-3 position of maytansinol is especially preferred. While the synthesis of maytansinol esters having a linking moiety is described in terms of linking portions containing disulfide bond, anyone skilled in the art will understand that the linking moieties with other chemical bonds (as described with above) may also be used with the present invention, as may be other maytansinoids. Specific examples of other chemical linkages include labile acid linkages, forlabile linkages, labile peptidase linkages, and labile esterase linkages. The disclosure of Is US Pat. No. 5,208,020, incorporated herein, teaches the production of maytansinoids having said linkages. The synthesis of maytansinoids and maytansinoid derivatives that have a disulfide moiety holding a reactive group are described in U.S. Patent Nos. 6,441,163 and 6,333,410, and U.S. Application No. 10 / 161,651, each of which is incorporated herein by reference. The maytansinoids containing reactive group, such as DMI, are reacted with an antibody, such as the DS6 antibody, in order to produce cytotoxic conjugates. These conjugates can be purified by means of HPLC or through gel filtration. Many excellent schemes for producing said antibody-maytansinoid conjugates are provided in U.S. Patent No. 6,333,410, and U.S. Requests Nos. 09 / 867,598, 10 / 161,651 and 10 / 024,290, each one of which is incorporated herein in its entirety. In general, a solution of an antibody in aqueous pH regulator can be incubated with a molar excess of maytansinoids having a disulfide moiety that has a reactive group. The reaction mixture can be quenched by the addition of excess amine (such as ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration. The number of maytansinoid molecules per antibody molecule can be determined by measuring spectrophotometric the ratio of the absorbance at 252 nm and 280 nm. An average of 1-10 molecules of maytansinoid / antibody molecules is preferred. Antibody conjugates with maytansinoid groups can be evaluated for their ability to suppress the proliferation of several undesirable cell lines in vitro. For example, cell lines such as the human squamous cell carcinoma line A-431, the human small cell lung cancer cell line SW2, the human breast tumor line SKBR3 and the Burkitt lymphoma line Namalwa can be used with ease for the determination of the cytotoxicity of these compounds. The cells to be evaluated can be exposed to the compounds for 24 hours and the remaining fractions of cells measured in direct assays by known methods. The IC50 values can then be calculated from the results of the tests.

Link groups containing PEG Maytansinoids may also be linked to cell binding agents using linking groups containing PEG, as set forth in United States of America Application No. 10 / 024,290. These PEG linkage groups are soluble in both water and non-aqueous solvents, and can be used for one or more cytotoxic agents to a cell-binding agent.

Exemplary PEG linking groups include hetero-bifunctional PEG linkers that bind to cytotoxic agents and cell binding agents at opposite ends of the linkers through a sulhydryl functional group or disulfide at one end, and an active ester at the other end . As a general example of the synthesis of a cytotoxic conjugate using a PEG linking group, reference is again made to US Application No. 10 / 024,290 for specific details. The synthesis starts with the reaction of one or more cytotoxic agents that have a reactive PEG portion with a cell-binding agent, resulting in the displacement of the terminal active ester from each reactive PEG portion by means of an amino acid residue of the reactive agent. cell linkage, to produce a cytotoxic conjugate comprising one or more cytotoxic agents covalently linked to a cell binding agent through a PEG linking group.

Taxans The cytotoxic agent used in the cytotoxic conjugates according to the present invention can also be a taxane or derivative thereof. The taxanes are a family of compounds that include paclitaxel (Taxol), a natural cytotoxic product and docetaxel (Taxotere), a semi-synthetic derivative, two compounds that are widely used in the treatment of cancer. The taxanes are mitotic-axis poisons that inhibit the depolymerization of tubulin, which results in cell death. While 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. In addition, compounds such as paclitaxel and docetaxel themselves are not potent enough to be employed in conjugates of cell-binding agents. A preferred taxane for use in the preparation of cytotoxic conjugates is the taxane of formula (XI): The methods for synthesizing taxanes that can be used in the cytotoxic conjugates of the present invention, together with the methods for conjugating the taxanes to cell binding agents such as antibodies, is described in detail in the US Patents. 5,416,064, 5,475,092, 6,340,701, 6,372,738 and 6,436,931, and in Requests of the United States of North America Nos. 10 / 024,290, 10 / 144,042, 10 / 207,814, 10 / 210,112 and 10 / 369,563.

Analogs CC-1065 The cytotoxic agent used in the cytotoxic conjugates according to the present invention can also be CC-1065 or a derivative thereof. CC-1065 is a potent anti-tumor antibiotic isolated from the culture broth of Streptomyces zelensis. CC-1065 is approximately 1000-fold more potent in vitro than commonly used anti-carcinogenic drugs, such as doxorubicin, methotrexate and vincristine (B.K. Bhuyan et al., Cancer Res., 42, 3532-3537 (1982)). CC-1065 and its analogs are described in U.S. Patent Nos. 6,372,738, 6,340,701, 5,846,545 and 5,585,499. The cytotoxic potential of CC-1065 has been correlated with its alkylation activity and its DNA-linkage or DNA-intercalation activity. These two activities reside in separate parts of the molecule. Thus, the alkylation activity is contained in the cyclopropapyrroloindole subunit (CPI) and the DNA binding activity resides in the two pyrroloindole subunits. Although CC-1065 possesses certain attractive characteristics as a cytotoxic agent, it has limitations in therapeutic use. Administration of CC-1065 to mice resulted in delayed hepatotoxicity that led to mortality on day 50 after an individual intravenous dose of 12.5 μg / kg. { V. L. Reynolds et al., J. Antibiotics, XXIX, 319-334 (1986)} . This has accelerated the efforts to develop analogs that do not cause delayed toxicity, and the synthesis of simpler analogues modeled in CC-1065 has been described. { M.A. Warpehoski et al., J. Med. Chem., 31, 590-603 (1988)} . In another series of analogs, the CPI portion was replaced by a cyclopropabenzindole (CB1) 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 the high potency of the original drug in vitro, without causing delayed toxicity in mice. Like CC-1065, these compounds are alkylation agents that bind to the lower DNA channel in a covalent manner to cause cell death. However, the clinical evaluation of the most promising analogs, Adozelesin and Carzelesin, has led to disappointing results. { B.F. Foster et al., Investigational New Drugs, 13, 321-326 (1996); I. Wolff et al., Clin. Cancer Res., 2.1717-1723 (1996)} . These drugs exhibit scarce therapeutic effects due to their high systemic toxicity. The therapeutic efficacy of the CC-1065 analogs can be greatly improved by changing the in vivo distribution through the targeted delivery to the tumor site, resulting in lower toxicity for normal tissues, and therefore, less systemic toxicity. In order to achieve this goal, conjugates of analogs and derivatives of CC-1065 have been described with cell binding agents that specifically target tumor cells. { Patents of the United States of North America; 5,475,092; 5,585,499; 5,846,545} . These conjugates commonly exhibit high in vitro target-specific cytotoxicity, and exceptional anti-tumor activity in human tumor xenograft modles in mice. { R.V. J. Chari et al., Cancer Res., 55, 4079-4084 (1995)} . Methods for synthesizing CC-1065 analogs that can be used in the cytotoxic conjugates of the present invention, together with methods for conjugating analogs for cell binding agents such as antibodies, are described in detail in U.S. Pat. of North America Nos. 5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618 in US Requests Nos. 10 / 116,053 and 10 / 265,452.

Other Drugs Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulisin and tubulisin analogs, duocarmycin and duocarmycin analogues, dolastatin and dolastatin analogues are also suitable for the preparation of conjugates of the present invention. The drug molecules can also be linked to the antibody molecules through an intermediate carrier molecule such as serum albumin. The compounds of Doxarubicin and Danorubicin, as described, for example, in U.S. Serial No. 09/740991, they are also useful cytotoxic agents.

Therapeutic composition The present invention also provides a therapeutic composition comprising: (a) an effective amount of one or more cytotoxic conjugates, and (b) a pharmaceutically acceptable vehicle. Similarly, the present invention provides a method for inhibiting the growth of selected cell populations comprising contacting the target cells, or tissue containing the target cells with an effective amount of a cytotoxic conjugate, or therapeutic agent comprising a cytotoxic conjugate, either alone or in combination with other cytotoxic or therapeutic agents. The present invention also comprises a method for treating a subject having cancer using the therapeutic composition of the present invention. The cytotoxic conjugates can be evaluated for in vitro potency and specificity through the methods described previously (see, for example, R.V.J. Chari et al, Cancer Res. 55: 4079-4084 (1995)). Anti-tumor activity can be evaluated in human xenograft models in mice through the methods described above (see, for example, Liu et al, Proc Nati Acad Sci 93: 8618-8623 (1996)) .

Suitable pharmaceutically acceptable carriers with well known and can be determined by those with ordinary skill in the art as warranties of clinical situation. As used in the presnete, vehicles include diluents and excipients. Examples of suitable carriers, diluents and / or excipients include: (1) pH regulated saline solution with Dulbecco's phosphate, pH 7.4, containing or not approximately 1 mg / ml to 25 mg / ml human serum albumin, (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)), and (3) 5% (w / v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20. The method for inhibiting the growth of selected cell populations can be practiced in vitro, in vivo, or ex vivo. As used herein, inhibiting growth means slowing the growth of a cell, decreasing cell viability, causing the death of a cell, lysing a cell and inducing cell death, whether for a short or long period. Examples of in vitro uses include autologous bone marrow treatments before transplantation within the same patient in order to eliminate diseased or malignant cells; bone marrow treatments before transplantation to eliminate the competent T cells and avoid graft-contraceptive disease (GVHD); cell culture treatments to remove all cells except the desired variants that do not express the target antigen; or eliminate variables that express undesirable antigen. The conditions of in vitro non-clinical use are easily determined by someone with experience in the technique. Examples of ex vivo clinic use are the removal of tumor cells or lymphoid cells from the bone marrow before autologous transplantation in the treatment of cancer or in the treatment of autoimmune disease, or to remove T cells and other lymphoid cells from autologous bone marrow. or allogeneic or tissues before transplantation in order to avoid engraftment graft versus receptor (GVHD). The treatment can be carried out as follows. The bone marrow is cultured from the patient or another individual and then incubated in a medium containing serum to which the cytotoxic agent of the invention is added. Concentrations range from about 10 μM to 1 pM, from about 30 minutes to about 48 hours at about 37 ° C. The exact conditions of concentration and the incubation time, i.e., the dose, are easily determined by one of ordinary skill in the art. After incubation the bone marrow cells are washed with the medium containing serum and returned to the patient through i.v. infusion. according to known methods. In circumstances where the patient receives another treatment such as a course of ablative chemotherapy or complete body irradiation between the time of the marrow culture and the reinfusion of the cells treated, the treated marrow cells are frozen and stored in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the cytotoxic conjugate of the invention will be supplied as solutions that are tested for sterility and for endotoxin levels. Examples of suitable protocols for administering the cytotoxic conjugate are as follows. The conjugates are supplied for 4 weeks as a bolus i.v. weekly. Bolus doses are supplied in 50 to 100 ml of normal saline to which 5 to 10 ml of human serum albumin can be added. The doses will be from 10 μg to 100 mg per administration, i.v. (range of 100 ng up to 1 mg / kg per day). More preferably, the doses will vary from 50 μg to 30 mg. More preferably, the doses will vary from 1 mg to 20 mg. After four weeks of treatment, the patient can continue to receive the treatment on a weekly basis. The specific clinical protocols with respect to the route of administration, excipients, diluents, doses, schedules, etc., can be determined by someone with ordinary experience in the technique as guaranteed by the clinical situation. Examples of medical conditions that can be treated according to in vivo or ex vivo methods to eliminate selected cell populations include malignancy of any type including, for example, lung, breast, colon, prostate, kidney, pancreas, ovaries, cervix cancer and lymphatic organs, osteosarcoma, synovial carcinoma, a sarcoma or a carcinoma in which it is expressed CA6, and other cas yet to be determined in which the CA6 glycoprotein is predominantly expressed; autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, rejection of heart transplant, and rejection of bone marrow transplantation; graft versus recipient disease; viral infections, such as mV infection, HIV infection, AIDS, etc .; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by someone with ordinary skill in the art.

Equipment The present invention also includes equipment, for example, comprising a described cytotoxic conjugate and instructions for the use of the cytotoxic conjugate to remove particular cell types. The instructions may include directions for using the cytotoxic conjugates in vitro, in vivo or ex vivo. In common, the equipment will have a compartment containing the cytotoxic conjugate. The cytotoxic conjugate may be in a lyophilized form, liquid form, or other form susceptible to being included in a kit. The equipment may contain additional I elements necessary to carry out the method described in the instructions on the equipment, such as a sterilized solution. to reconstitute a lyophilized powder, additional agents for combination with the cytotoxic conjugate before administration to a patient and tools that help in the administration of the conjugate to a patient.

Additional Modalities The present invention further provides monoclonal antibodies, humanized antibodies and epitope binding fragments thereof which are also labeled for juso in diagnostic research applications. In preferred embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent, or a metal ion. A diagnostic method is also provided in which the labeled antibodies or epitope binding fragments thereof are administered to a subject presumed to have ca, and the distribution of the tag within the body of the subject is measured or monitored.

Examples The broad scope of this invention is best understood with refereto the following examples, which are not intended to limit the scope of the invention to the specific embodiments.

Example 1: Identification of positive antigenic and negative antigenic cell lines by flow cytometry binding assays.

Flow cytometric analyzes were used to locate the epitope DS6, CA6, for the cell surface. Human cell lines were obtained from the American Type Culture Collection (ATCC) with the exception of OVCAR5 cells (Kearse et al., Int. J. Ca 88 (6): 866-872 (2000)), OVCAR8 and IGROVI ( M. Seiden, Massachusetts General Hospital). All cells were cultured in RPMI 1640 supplemented with 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin (Cambrex Bio Scie Rockland, ME) and 10% v / v fetal bovine serum (Atlas Biologicals, Fort Collins, CO), referred to in the following as the culture medium. The cells were maintained at 37 ° C, 5% CO2 humidified incubator. The cells (1-2 x 1? 5 cells / well) were incubated, on ice for 3-4 h, with serially diluted cotrations of the DS6 antibody prepared in pH regulator FACS (2% goat serum, RPMI) In 96-well plates, the cells were rotated in a tabletop centrifuge at 1500 rpm for 5 minutes at 4 ° C. After the medium was removed, the wells were filled with 150 μl of pH-regulator FACS. The washing step was repeated, goat anti-mouse FITC-labeled IgG (Jackson Immunoresearch) was diluted 1: 100 for the pH regulator FACS and incubated with the cells for 1 hour on ice. a thin sheet to avoid the whitewashing of the signal. After two washes, the cells were fixed with 1% formaldehyde and analyzed in a flow cytometer. Predominantly, the CA6 epitope was found in cell lines of ovarian, breast, cervical and pancreatic origin (Table 3) as predicted from tumor immunohistochemistry. However, some cell lines of other tumor types exhibited limited expression of CA6. The DS6 antibody binds to an apparent KD of 135.6 pM (in PC-3 cells, Table 3). The maximum mean fluorescence (Cuasdro 3) of the binding curves (Figure 1) in the positive antigen cell lines are suggestive of the relative antigen density. Table 3 Kd Line Kd Line Cellular Tissue Antigen MMF 'Apparent (M) Fabric Cell Antigen MMF' Apparent (M) Blood HL-60 - Caov-3 Ovary + 46520 5478x10 o9 Jurkat Sanguineous - Caov-4 Ovary + 149004043 x10 o9 Namalwa Sanguineous - ES-2 Ovary - U-937 Sanguineous - IGROVI Ovary - Brain T98G + 3594 1775x10 '° OV-90 Ovary - BT-20 Breast + 232209142x10"10 OVCAR-3 Ovary - BT-474 Breast - OVCAR5 Ovary + 97101 473 x10 o9 BT-483 Seno + 191100 1366x108 OVCAR8 Ovary - BT-549 Seno + 7139 1046 x I 0 M PA-I Ovary - BED-I Seno + 1246 2330 x1o "09 SK-OV-3 Ovary - MCF-7 Seno + 8141 2890 x10 o9 SW626 Ovary - MDA-MB-157Sen + 8635 1972 10 0 TOV-112D Ovary - MDA-MB-231 Sine + 3185 1460 x10 o9 TOV-21G Ovary + 8779 306 x10 0 MDA-MB-468 Breast + 71588 127 xlO 0 AsPC-l Pancreas - SK-BR-3 Breast - BxPC-3 Pancreas + 7999 5263 x10 o9 T-47D Seno + 55958 3424 x10o9 HPAC Pancreas + 222800 2348 x 10 ZR-75-1 Seno + 81167 4299 x10 o9 HPAF-II Pancreas + 26650 2 81 1 x 1009 HeLa Cervix + 24250 6938x1010 Hs766T Pancreas + 18290 2319 x 10 KB Cervix + 11956 1.110x10o9 MIAPaCa2 Pancreas. WISH Cervix + 113355 2380 x10 o9 MPanc96 Pancreas - Colo205 Colon - SU 8686 Pancreas + 3686 1043X10'09 DLD-I Colon - SW 1990 Pancreas + 3617 3679x10-10 HCT-8 Colon - PC-3 Prostate + 2481 1356x10 '° HT-29 Colon - A375 Skin - Caki-1 Kidney - SKMEL28 Skin - A549 Lung - KLE Uterus - SW2 Lung - mean average maximum relative fluorescence Example 2: Characterization of DS6 Epitope The properties of antigen DS6, CA6, were analyzed by means of a spot staining of CA6-positive cell lysates (Caov-3) that were digested with proteolytic treatments (pronase and proteinase K) and / or glycolytic (neuraminidase and acid Newspaper). For positive controls, other antibodies that recognize a variety of epitope types were tested on lysates of antigen positive cell lines (Caov-3 and CMI, Colo205 and C242, SKMEL28 and R24). CMI is an antibody that recognizes a propetin epitope from the variable number tandem repeat domain (VNTR) of Muc-1 and therefore provides a control for a protein epitope. C242 binds to a specific scleral acid-dependent glycogen of novel colorectal cancer in Muc-1 (CanAg) that provides a control for a glycopod on a protein. R24 binds to ganglioside GD3 that is melanoma specific and therefore provides a control for a glycopod on a non-protein support. Caov-3, Colo205, and SKMEL28 cells were placed on plates in 15 cm tissue culture plates. Culture media (30 mL / plate) were renewed the day before lysis. A modified RIPA pH regulator (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.25% sodium deoxycholate), protease inhibitors (PMSF, Pepstatin A, Leupeptin, and Aprotinin), and PBS were pre-cooled on ice. After the medium of culture was aspirated from the plates, the cells were washed twice with 10 ml of cooled PBS. All subsequent stages were conducted on ice and / or cooled to 4 ° C. After the last wash the PBS was aspirated, the cells were lysed in 1-2 mL of lysis buffer (pH regulator RIPA with newly added protease inhibitors up to a final concentration of 1 mM PMSF, 1 μM Pepstatin A, 10 μg / ml Leupeptin, and 2 μg / ml Aprotinin). The lysates were removed from the plates using a cell transporter and crushed by pipetting the suspensions up and down (5-10 times) with an 18G needle. The lysates were rotated for 10 minutes and then centrifuged in a microcentrifuge at maximum (13K rpm) for 10 min. The pellets were discarded and the supernatants were then tested using a Bradford Protein assay kit (Biorad). The lysates (2 μl) were pipetted directly onto 0.2 μm dry nitrocellulose membranes. The spots were allowed to air dry for approximately 30 minutes. The membrane was sectioned into pieces each containing an individual point. The spots were incubated in the presence of pronase (1 mg / ml enzyme, 50 mM Tris pH 7.5, 5 mM CaCl2), proteinase K (1 mg / ml enzyme, 50 mM Tris pH 7.5, 5 mM CaCl2), neuraminidase (20 mU / ml enzyme, 50 mM sodium acetamide pH 5.5, 5 mM CaCI2, 100 μg / ml BSA) or periodic acid (20 mM, 0.5 M sodium acetate pH 5) for 1 hour at 37 ° C. the reagents were purchased from Roche (enzymes) and VWR (periodic acid). The membranes were washed (5 minutes) in washing pH regulator T-TBS (0.1% Tween 20, IX TBS), blocked in blocking buffer (3% BSA, T-TBS) for 2 hours at room temperature, and incubated overnight with 2 μg / ml of primary antibody (ie, DS6, CMI, C242, R24) in a blocking pH regulator at 4 ° C. The membranes were washed three times for 5 minutes in T-TBS and then incubated in HRP-conjugated goat anti-mouse (or human) secondary antibody IgG (Jackson Immunoresearch; 1: 2000 dilution in blocking buffer) for 1 hour at room temperature. Immunostains were washed three times and developed using an ECL system (Amersham). The immunostains (FIG. 2) of the digested control lysates showed that the CMI signal was destroyed by the proteolytic treatments while the signals of the glycolic digests were not affected as would be expected for an antibody that recognizes a protein epitope. The C242 signal was destroyed by the proteolytic or glycolytic treatments as would be expected for an antibody recognizing a glycogen found in a protein. The signal R24 signal, not affected by the proteolytic treatments, was abolished with neuraminidase or periodate treatments as expected for an antibody that recognizes a ganglioside. DS6 immunostaining of spot stains of digested Caov-3 lysate showed signal loss to treatment with proteolytic and glycolytic compounds. Therefore, just like C242, DS6 binds to an epitope carbohydrate in a proteinaceous core. In addition, the signal in the DS6 immunostaining was sensitive to treatment with neuraminidase. Therefore, CA6, like CanAg, is a glycapto dependent on sialic acid. In order to confirm the nature of the CA6 carbohydrate, the Caov-3 lysate was located on the PVDF membrane and treated with the chemical deglycosylating agent, trifluoromethane sulfonic acid (TFMSA), under nitrogen at room temperature for 5 minutes. The staining was washed with T-TBS and immunostained with CMI or DS6 (Figure 3). The DS6 signal was destroyed upon acid treatment providing additional evidence that CA6 is a glycopod. The improvement of the CMI signal to the TFSMA treatment indicates that the acid treatment did not affect the protein in the filter and suggests that glycolytic treatment unmasked the protein epitope recognized by CMI. To further determine the structure of the carbohydrate in which CA6 resides, spot stains were digested with N-glycanase, O-glycanase, and / or sialidase (Figure 4). Caov-3 cell lysates (100 μg, 30 μl) were incubated at 100 ° C for 5 minutes with 2.5 μl denaturing buffer (Glyko) containing SDS and β-mercaptoethanol. The denatured lysates were then digested with 1 μl of N-glycanase, O-glycanase, and / or Sialidase A (Glyko) at 37 ° C for 1 h. The digested lysates were localized (2 μl) on nitrocellulose and immunostained as described above.

N-glycanase had no apparent effect on DS6 immunostaining signals. However, samples digested with sialidase produced no signal. Because O-glycanase was unable to digest sialylated O-linked carbohydrates without prior treatment with sialidase, the DS6 signal from samples processed with O-glycanase alone would not be affected. N-glycanase, in contrast, does not require pretreatment with glycosidic enzymes for activity. The fact that treatment with N-glycanase does not affect the DS6 signal suggests that the CA6 epitope is most likely present in O-linked carbohydrate chains sialylated.

Example 3: Determination of the antigen on which the CA6 epitope is located To identify the antigen on which the CA6 sialogliquetope is located, DS 6 immunoprecipitates were analyzed by SDS-PAGE and Western staining. Supernatants of cell lysate (1 mL / sample, 3-5 mg protein) were pre-rinsed with Protein G beds (30 μL), equilibrated with 1 mL of RJPA buffer, for 1-2 hours, with rotation, at 4 ° C. All subsequent stages were conducted on ice and / or in a room cooled to 4 ° C. The pre-rinsed beds were briefly rotated (2-3 s) in a microcentrifuge. The pre-cleared supernatants were transferred to new tubes and incubated during the night with 2 μg of DS6, with rotation. The new, equilibrated Protein G beds (30 μl) were subjected to lysates and incubated for 1 hour, with rotation. The bedded-lysate suspensions were briefly rotated in a microcentrifuge and samples of the post-immunoprecipitation lysates were optionally taken. The beds were washed 5-10 times with 1 mL of RIPA regulator. Immunoprecipitated DS6 samples were then digested with 30 μl neuraminidase (20 mU neuraminidase (Roche), 50 mM sodium acertate pH 5.5, 5 mM CaCI2, 100 μg / ml BSA) or 30 μl periodic acid (20 mM periodic acid (VWR ), 0.5M sodium acetate pH 5) for 1 hour at 37 ° C. Then furon were suspended again in 30 μl of 2X sample charge regulator (containing β-mercaptoethanol). The beds were boiled for 5 minutes and the supernatants of the charge regulator were loaded in 4-12% or 4-20% Tris-Glycine gels (Invitrogen). The gels were operated in Laemmli electrophoresis operation regulator at 125 V for 1.5 h. The gel samples were transferred, overnight at 20 mA, onto 0.2 μm nitrocellulose membranes (Invitrogen) using a Mini Trans-blot transfer apparatus (Biorad). The membranes were immunostained with DS6 as described above in Example 2. Alternatively, the immunoprecipitated beds were first denatured and then digested enzymatically with N-glycanase, O-glycanase and / or sialidase A (Glyko). The beds were suspended again in 27 μl of incubation buffer and 2 μl of denaturing solution (Glyko) and incubated at 100 ° C for 5 minutes. After cooling to room temperature, detergent solution (2 μl) was added and the samples were incubated with 1 μl of N-glycanase, O-glycanase, and / or Sialidase A at 37 ° C for 4 hours. After adding 5X sample charge regulator (7 μl), the samples were placed at boiling for 5 minutes. The samples were subjected to SDS-PAGE and immunostained as described above. DS6 immunoprecipitates a > 250 kDa protein band that can be seen in positive antigen cell lysates (Figure 5A, B, and C). In some of the cell lines (ie, T-47D), a doublet was observed. The band > 250 kDa was abolished in Caov-3 immunoprecipitates that were treated with neuraminidase or periodic acid (Figure 5 A and B) suggesting that the CA6 epitope resides in the > 250 kDa. It was also shown that the band > 250 kDa is insensitive to the N-glycanase treatment of immunoprecipitates consisting of CA6 residing in an O-linked carbohydrate (Figure 5F). It is further argued that the 250 kDa band is the CA6 antigen is the fact that DS6 does not immunoprecipitate said band from the negative antigen DS6 cells (Figure 5D and E). Several lines of evidence suggested that the CA6 antigen was Mucl. Due to the high molecular weight and sensitivity for the specific enzymes of O-linked carbohydrate, it seemed also that the CA6 antigen was a mucin. The overexpression of mucin is well characterized in tumors, particularly of the breast and ovary, consisting of the main DS6 tumor reactivities. In addition, CA6, like CanAg (a sialogliquetopo in Mucl), is not susceptible to perchloric acid precipitation suggesting that the CA6 antigen is strongly O-glycosylated. The observation that in some cell lines expressing DS6, DS6 immunoprecipitated a doublet of > 250 kDa suggested that the CA6 was Mucl. A hallmark of Mucl in humans is the presence of two distinct Mucl alleles that differ in the number of tandem repeats resulting in the expression of two Mucl proteins of different molecular weights. To test whether CA6 was found in Mucl, the DS6 immunoprecipitates from the Caov-3 lysate were subjected to SDS-PAGE and immunostained with DS6 or an Mucl VNTR antibody, CMI. As you can see in Figure 6A, CMI reacts strongly with the >band250 kDa immunoprecipitated by DS6. In Figure 6B, the immunoprecipitates DS6 and CMI immunoprecipitates of the HeLa cell lysate show the same doublet > 250 kDa when immunostained with DS6 or CMI. These results indicate that the CA6 epitope is in fact unicapped in the Muc-1 protein. The DS6 doublet observed in HeLa cells (and T-47D) can be explained by the fact that Muc-1 expression is driven by different alleles that have different tandem repeats. Although CMI and DS6 bind to the same Muc-1 protein, they are different epitopes. The chemical deglycosylation of the dot ticnion of the Caov-3 lysate by means of trifluoromethane sulfonic acid (TFMSA) abolished the DS6 signal (Figure 3). However, this same treatment improved the CMI signal. The deglycosylation may have revealed hidden epitopes for the CMI antibody. In addition, a comparison of the results of the flow cytometry linkage of DS6 and CMI (Table 4) demonstrates that the CA6 epitope does not exist in every cell expressing Mucl. It is interesting to note that the CA6 epitope is not expressed in Colo205 (Table 3), a concoded cell line expressing high levels of the Mucl CanAg sialoglicatope.

Table 4 DS6 CMI Line Kd (M) Kd (M) Cellular MMF * Apparent MMF Apparent BT549 71.39 1.046 xl? "187.90 6.056 x 10 ° CaOV3 465.20 5.478 x 10 1031.00 7.479 x 10" DS6 positive CMI positive HeLa 242.50 6 938 x 10"10 334.80 2,907 X IO Kb 119.56 1 110 x 10" 09 338.00 5 345 10"MCF7 81.41 2,890 x 10 ° 9 1023.00 8,694 10 ° KLE 27.48 561.70 8.156 10 'DS6 negative OVCAR3 21.19 19250 5 940 10" CIM positive SKOV3 17.53 49.41 6.246 10 MMF = maximum mean relative fluorescence Example 4: Quantitative analysis of detached CA6 epitope Because the CA6 epitope resides in Mucl, a molecule known to break off within the bloodstream in many cancer patients, a quantitative approach was taken to determine if such levels would be prohibitive for a DS6 antibody therapy. It is thought that the binding of the circulating antibody to the antigen leads to the rapid release of immune complexes within the blood. If a significant portion of the dose of antibody administered is rapidly removed from the circulation, it is likely that the amount reaching the tumor is decreased resulting in reduced antitumor activity of a therapeutic antibody. When the antibody is conjugated to a high potency cytotoxic compound the rapid release of the conjugate could potentially increase non-specific toxicity. Therefore, in the case of small drug-antibody conjugates such as DS6-DM1, high levels of dispersed antigen can be expected to reduce the anti-tumor effect and increase the dose-limiting toxicity. Recent clinical trials of therapeutic antibodies have provided information on the impact of distributed antigen concentration on pharmacokinetics. For example, in clinical trials with trastuzumab (Herceptin), an antibody used for the treatment of mestastatic breast cancer expressing her2 / neu, it was shown that the pharmacokinetics of trastuzumab release remains unchanged when the level of Her2 / neu distributed was lower than 500 ng / mL (Pegram et al., J. Clin Oncol 16 (8): 2659-71 (1998) Assuming a molecular weight of Her2 / neu distributed of 110,000 Daltons, a molar concentration distribution of Her2 / neu below 4.5 nM seems to have little influence on pharmacokinetics. In another example, a clinical trial with cantuzumab mertansine (huC242-DMI) indicated that there is no correlation with the levels of distributed CanAg pretreatment (epitope C242) and the pharmacokinetics of antibody release (Tolcher et al., J. Clin. Oncol 21 (2): 211-22 (2003) .The CanAg epitope, similar to the CA6 epitope recognized by DS6, is a tumor-specific O-linked sialoglicatopo, unique in Mucl, however, the heterogeneous nature of the CanAg epitope makes it difficult to quantification in molar terms In the general population the Mucl alleles vary in length depending on the number of tandem repeats in the variable number tandem repeat domain (VNTR) Several O-linked glycosylation sites are presented in each tandem repeat. the complexity of the CanAg expression is the cell-to-cell variation in the inherent glycosyltransferase activity.Therefore, a large range of CanAg epitopes per molecule is possible. the Mucl even in a single patient. further, the ratio of CanAg epitope per molecule Mucl will be different across the population of patients. For this reason, CanAg distributed in serum samples is measured through interspersed ELISA where Mucl distributed with CanAg epitope is captured by C242 and detected by a C242 / Streptavidin HRP stained system. The distributed CanAg is quantified in standardized units (U) proportional to the number of epitopes per ml of serum instead of a molar concentration of Mucl. By analogy, a similar situation occurs for the quantification of CA6 epitopes distributed. In contrast, for trastuzumab there is only one epitope per molecule of her2 / neu distributed, greatly satisfying the quantification of the distributed antigen. To relate the distributed epitope levels CA6 with those found in clinical trials with trastuzumab and cantuzumab mertansine, a method was developed to obtain molar concentrations of complex distributed epitopes such as sialogliquetopos in Mucl. First, a simple intercalated ELISA assay for DS6 was established. A representation of the test is shown in Figure 7A. DS6 was used to capture Mucl that has CA6 epitope. Because each Mucl molecule has multiple CA6s Epitope, biotinylated DS6 was used as the trace antibody. The biotinylated DS5 bound to the captured CA6 was detected by means of Streptavidin-HRP using ABTS as the substrate. The CA6 epitope was captured from the serum of a patient with ovarian cancer or from standards that come from a commercially available Mucl test kit (CAI 5-3) used to monitor distributed Mucl in patients with breast cancer. Units / ml of DS6 were set arbitrarily the same for standard units / ml of CAI 5-3. Figure 7B shows the results of the DS6 interspersed ELISA test in which the CAI 5-3 standards were used. The curve generated is very similar to auqella obtained with CAI 5-3 standards in the CAI 5-3 test. In order to convert the unit / ml DS6 to a molar concentration of CA6, a standard curve was required for DS6 biotinila or that converts the signal to picograms of DS6. Assuming a 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 mols of CA6 captured per volume of aggregate sample can be calculated. In Figure 8 A and B are the representations of two alternative media to generate a standard curve for biotinylated DS6. In Figure 8A, the Goat anti-mouse IgG monoclonal antibody was used to capture the biotinylated DS6 which in turn is detected in a manner identical to that used in the interspersed ELISA assay shown in Figure 7. In the method shown in FIG. Figure 8B DS6 biotinylated DS6 is plated directly onto the ELISA plate and detected as in Figure 8A. As seen in Figure 8C, the standard curves of biotinylated DS6 generated by each method are in concordance. Table 5 shows the analysis of suerp samples from patients with ovarian cancer for different distributed antigens. CAI 25 ELISA is usually used to monitor the treatment of patients with ovarian cancer by measuring units / ml of distributed CA125. The CA125 status was provided with the serum samples. CAI 5-3 ELISA is generally used to monitor the treatment of patients with breast cancer by measuring the units / ml of Mucl distributed using capture antibodies and detections that recognize epitopes other than those recognized by DS6. In Table 5, CAI 5-3 is measured in serum samples from patient with ovarian cancer. Table 5 Serum CAI 251 CAI 5-3 'DS62 DS6' DS6 No. (U / ml) (U / ml) (U / ml) (pM) (pM) 4 72.80 117.72 29.79 52.13 188.94 5 3651.90 98.19 567.02 654.44 > 2560.00 6 930.50 87.08 504.15 667.56 2505.00 7 76.00 72.70 135.65 246.94 778.25 8 32.50 18.44 39.96 65.19 239.88 9 551.70 292.39 > 975.61 1512.31 > 2560.00 10 90.00 42.40 485.06 13 197.50 20.61 35.92 61.06 216.25 14 100.60 6.13 12.39 23.19 88.06 15 34.60 59.18 199.85 286.63 1228.56 17 196.40 56.75 66.53 130.44 405.88 18 16.90 30.45 34.43 60.81 223.69 19 22.00 263.93 118.98 191.69 728.94 22 110.70 21.44 16.46 29.94 111.38 determined by commercial ELISA equipment determined by commercial C A15-3 standards (1 CA15 -3 U = 1 DS6 U) standard curve goat anti-mouse IgG &biotin-DS6 standard curve biotin- DS6 For the CAI 5-3 values reported in Table 5, a CAI 5-3 Enzyme Assay Kit commercially available from CanAg Diagnostics was used. For units / ml of DS6, a standard curve was generated using the CAI 5-3 standards (from CanAg Diagnostics CAI 5-3 Enzyme Immunoassay equipment) in DS6 interspersed ELISA. Units / ml DS6 were arbitrarily set equal to CAI 5-3 units / ml. In the last two picomolar columns (pM) distributed CA6 was calculated using the standard biotinylated CA6 curves shown in Figure 8C. For the quantitative analysis of CanAg serum levels, CanAg serum levels were those reported for patients participating in a clinical trial of cantuzumab mertansine before treatment (Tolcher et al., J. Clin Oncol 21 (2): 211-22 (2003) .An ELISA test analogous to that described for DS6 was used to generate a CanAg standard curve using CanAg standards C242 was used to capture the CanAg standards.The detection of captured CanAg was achieved using a biotinylated C242 tracer followed by development with streptavidin-HRP using ABTS as substrate.A standard curve of C242-biotinylated was prepared as was done for biotinylated DS6 allowing the conversion of units / ml to a molar concentration of circulating CanAg epitopes In Table 6. CanAg levels from clinical trial patients of cantuzumab mertansine were reported together with the corresponding calculated molar concentrations of Circulating CanAg.

Table 6 CanAg1 CanAg2 CanAg3 .U / ml) pMM) 31240 19185.7 34592.8 8687 3535 9619.3 7456 4579 8256.2 3686 2263.7 4081.6 1447 888.7 1602.3 1262 775 1397.4 718 441 795.1 547 335.9 605.7 394 242 436.3 381 234 421.9 329 202.1 364.3 322 197.8 356.6 306 187 338.8 284 174.4 314.5 247 151.7 273.5 242 148.6 268 229 140.6 253.6 227 139.4 251.4 184 113 203.7 120 73.7 132.9 107 65.7 118.5 100 61.4 110.7 81 49.7 89.7 81 49.7 89.7 67 41.1 74.2 53 32.5 58.7 45 27.6 49.8 43 26.4 47.6 39 24 43.2 36 22.1 39.9 24 14.7 26.6 18 11.1 19.9 17 10.4 18.8 < 10 6.1 11.3 < 10 6.1 11.3 < 10 6.1 11.3 < 10 6. 1 11.3 levels of pre-treatment of circulating CanAg measured by ELISA intercalated 2 goat anti-rabbit IgG & biotin-curve standard C242 3 biotin-curve standard C242 A comparison of the p6 levels of CA6 distributed in ovarian cancer patients with those calculated for CanAg distributed in cancer patients showed that in general distributed CA6 levels are similar to the levels of distributed CanAg. Furthermore, only 2 of the 16 serum samples from patient with ovarian cancer potentially have CA6 levels greater than 4.5 nM, (serum samples 5 and 9 for which the signal was outside the range of the standard curve), level at which the pharmacokinetics of altered herceptin was observed in clinical trials with patients with Her2 / neu-positive breast cancer. CanAg levels above 4.5nM were only observed in 3 of the 37 clinical trial patients. In this clinical trial there was no correlation with the levels of CanAg distributed and the faster release of cantuzumab mertansina. However, the patient with the highest CanAg level (31240 U / ml) was only sampled during 8 hours post-transfusion. These results indicate that certain epitopes of Mucl, such as CA6 and CanAg, while it is distributed in cancer patients, are not at prohibitive levels for the therapeutic treatment of antibody.

Example 6: Cloning of Variable Regions of Murine DS6 Antibody.

Murine monoclonal antibodies such as DS6 have limited utility in a clinical setting because they are recognized as foreign by the human immune system. Patients rapidly develop human anti-mouse antibodies (HAMA) resulting in the rapid release of murine antibodies. For this reason, the variable region of murine DS6 (muDS6) was coated in order to produce humanized DS6 antibodies (huDS6). The variable regions of murine DS6 antibody were cloned by RT-PCR. Total RNA was purified from a confluent T175 flask of DS6 hybridoma cells using the Qiagen RNeasy miniprep kit. The RNA concentrations were determined through UV spectrophotometry and RT reactions were performed with 4-5 μg total RNA using the equipment Gibco Superscript II and random hexamer primers. PCR reactions were performed with degenerate primers based on those described in Wang Z et al., J Immunol Methods. Jan 13; 233 (1-2): 167-77 (2000). The RT reaction mixture was used directly to degenerate the PCR reactions. The 3 'light chain primer, HindKL, (TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC) (SEQ ID NO: 25) and the 3' heavy chain primer, BamlgGI, were used.

(GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC) (NO ID SEC: 26), and for the 5 'end PCR primers were Sac IMK (GGGAGCTCGAYATTGTGMTSACMCARWCTMCA) (NO SEC ID: 27) for the light chain and an equal mixture of EcoRIMHI (CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC) (SEQ ED NO: 28) and EcoRIMH2 (CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG) (NO SEC ID: 29) for the heavy chain (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). PCR reactions were standard except that they were supplemented with 10% DMSO (50μl reaction mixtures contained in the final concentrations of reaction regulator IX (ROCHE), 2mM each dNTP, ImM each primer, 2 μl RT reaction, 5 μl DMSO, and 0.5μl Taq (ROCHE)). PCR reactions were performed in an MJ research thermal cycler using a program adapted from Wang Z et al., (J Immunol Methods, Jan 13; 233 (1-2): 167-77 (2000)): 1) 94 ° C, 3 minutes; 2) 94 ° C, 15 seconds; 3) 45 ° C, 1 minute; 4) 72 ° C, 2 minutes; 5) cycle back to stage # 2 29 times; 6) finished with a final extension stage at 72 ° C for 10 minutes. The PCR products were cloned into pBluescript II SK + (Stratagene) using restriction enzymes created by the PCR primers. The Seqwright sequencing services sequenced the heavy and light chain clones. To confirm the 5 'end cDNA sequences, additional PCR and cloning was performed. The DS6 light chain and heavy chain cDNA sequences, determined from the degenerate PCR clones, were connected to the NCBFs Blast research site and the murine antibody sequences were stored with presented signal sequence. The PCR primers were designed from these signal peptides using the conserved tensions between the related DNA sequences. EcoRI restriction sites were added to the leader sequence primers (Table 7) and these were used in RT-PCR reactions as described above.

Several individual light and heavy chain clones were sequenced to identify and avoid possible sequence errors generated by polymerase. Only one individual sequence of the light chain and heavy chain RT-PCR clones was obtained. These sequences were sufficient to design primers that could amplify murine DS6 light and heavy chain sequences that extend within the signal sequence. Subsequent clones from these follow-up PCR reactions confirmed the 5 'end sequences of the variable region that had been altered by the original degenerate primers. Cumulative results from the different cDNA clones gave the final murine light chain and heavy DS6 sequences presented in Figure 9. Using the Kabat and AbM definitions, the three light chain and heavy chain CDRs were identified (Figures 9 and 9). 10). a search of the NCBI Ig Blast database indicates that the light chain variable region of the muDS6 antibody is most likely derived from the germline of the murine VK ap4 Ig gene, while the heavy chain variable region is most likely derived from the germ line of the murine IgVh J558.41 gene (Figure 11).

Example 7: Determination of Surface Residues of the Variable Region of the DS6 Antibody The antibody coating techniques described by Pedersen et al. (1994) and Roguska et al. (1996) starting with the prognosis of the surface residues of the murine antibody variable sequences. A surface residue is defined as an amino acid having at least 30% of its total surface area accessible to a water molecule. In the absence of a resolved structure to find the surface residues for muDS6, the ten antibodies were aligned with the most homologous sequences in the set of 127 antibody structure files (Figure 12). The accessibility of the solvent for each Kabat position was averaged for these aligned sequences (Figures 13A and B). The surface positions with average accessibilities between 25% and 35% were subjected to a second round of analysis by comparing a subset of antibodies containing two identical residues flanking each side (Figures 13A and B). After the second round analysis, the 21 surface residues predicted for the heavy chain of muDS6 were increased to 23, adding Tyr3 and Lys23 to the list of residues with predicted surface accessibility greater than 30%. In most coated antibodies the Kabat definition of the heavy chain CDR1 was used, although for DS6 the AbM definition was inadvertently used during the calculations so the T28 heavy chain redisium was not defined as a surface residue of structure as it would have been otherwise. The number of light chain surface positions is reduced from 16 to 15 because the surface accessibility of Ala80 was reduced from 30.5% to 27.8% in the second round analysis. Together, the variable sequences of light chain and heavy chain muDS6 possess 38 predictable surface structure residues predicted.

Example 8: Selection of human antibody The surface positions of the murine DS6 variable region were compared with the corresponding positions in human antibody sequences in the Kabat database (Johnson G, Wu TT, Nucleic Acids Res. Jan l; 29 (l): 205-6 ( 2001)). The SR antibody database management software (Searle, 1998) was used to extract and align the surface residues from natural human heavy and light chain antibody pairs. The variable region surface of human antibody with the most identical surface residues, with special consideration given to positions that come within 5 A of a CDR, was selected to replace the variable region surface residues of the murine DS6 antibody.

Example 9: Expression vector for guimeric and humanized antibodies The paired sequences of light and heavy chain were cloned into an individual mammalian expression vector. The PCR primers for the human variable sequences create restriction sites that allowed the human signal sequence to be added in the pBluescriptII cloning vector. The variable sequences could then be cloned into the mammalian expression plasmid with EcoRI and BsiWI or HindIII and Apal for the light chain or heavy chain respectively (Figure 14). The light chain variable sequences were cloned in structure over the human IgKappa constant region and the heavy chain variable sequences were cloned in the IgGammal human constant region sequence. In the final expression plasmids, the human CMV promoters drive the expression of the light chain and heavy chain cDNA sequences.

Example 10: Identification of Waste that Negatively Affects Activity DS6 In most humanizations to date, a molecular model of subject antibody has been developed to identify residues close to a CDR as potential problematic residues. With an expanding number of coated antibodies from which to work, historical experience is at least as effective in forecasting problems as it is in developing a model, so a molecular model for DS6 has not been built. . Instead, the Surface residues of murine DS6 were compared to those of previously coated antibodies and residues were identified with low to high risk to affect the binding activity of the antibody. Similar sets of residues are identified as being widely used to be within 5A of a CDR in both the resolved antibody structures available and in the molecular models from previous humanizations. Using these data, Table 1 provides the murine DS6 residues that are likely to be found near and possibly within 5 A of a CDR. Many of these positions have changed in previous humanizations, although only heavy chain position 74 has resulted in a loss of enalce activity. The murine residue was retained in this position in huC242 and huB4 in order to preserve the binding activity of the murine antibody. On the other hand, this same position was changed for the corresponding human residue in humanized 6.2G5C6 without loss of activity (6.2G5C6 is the anti-IGFI-R antibody frequently referred to simply as anti-C6). While any of the residues in Table 1 could present a problem in the humanized antibody, the P73 heavy chain residue will be of particular interest due to the experiences anticipated in this position.

Example 11; Selection of the most Homologous Human Surface The candidate human antibody surfaces for coating muDS6 were extracted from the Kabat antibody sequence database using SR software. This software provides an interface to search only specified residue positions against the antibody database. In order to preserve the natural pairs, the surface residues of both light and heavy chains were compared together. The most homologous human surfaces from the Kabat database were aligned for classification of the sequence identity. The top 3 surfaces as they are aligned by the SR Kabat database software are provided in Table 2. The surfaces were then compared to identify which human surfaces would require the latest changes for the residues identified in Table 1. The anti-antibody -Rh (D), 28E4 (Boucher et al, 1997), requires the last number of surface waste changes (11 total) and only 3 of these residues are included in the list of potential problematic residues. Since the 28E4 antibody provides the most homologous human surface, it is the best candidate to coat muDS6.

Example 12: Construction of DNA sequences for humanized DS6 Antibodies The 11 surface residue changes for DS6 were made using PCR mutagenesis. PCR mutagenesis was performed in the cDNA clones from the murine DS6 variable region to produce the human DS6 coated gene. The humanization primer sets were designed to effect the amino acid changes required for the coated DS6, shown below in Table 8.

Table 8 PCR reactions were standard except that they were supplemented with 10% DMSO (50 μl reaction mixtures contained final concentrations of reaction regulator IX (ROCHE), 2 rriM each dNTP, 1 mM each primer, 100 ng template, 5 μl DMSO, and 0.5 μl Taq (ROCHE)). They were operated in a MJ Research thermocycler with the following program: 1) 94 ° C, 1 minute; 2) 94 ° C, 15 seconds 3) 55 ° C, 1 minute; 4) 72 ° C, 1 minute; 5) return cycle to stage # 2 29 times; 6) finish with a stage of final extension at 72 ° C for 4 minutes. The PCR products were digested with their corresponding restriction enzymes and cloned in Bluescript cloning vecteres. The clones were sequenced to confirm the amino acid changes. Since the change of the residue of heavy chain residue P73 has caused problems in the past, two versions of the heavy chain were built, one with the human 28E4 T73 and one that retains the P73 of murine. The other 10 surface residues were changed from the murine residue to the human 28E4 residue in both versions of humanized DS6 (Table 2). According to the usual nomenclature method, the most human version is 1.0 since it has 11 human surface residues. The heavy chain version that retains the murine P73 is named version 1.2 in case more versions are required so version 1.1 is reserved for a version that contains the maximum number of murine residues. The amino acid sequences of the two humanized versions are shown to be aligned with the amino acid sequence of murine DS6 in Figure 15A and B. The humanized DS6 antibody genes were cloned into the antibody expression plasmid (Figure 14) for transient transfections and stable The cDNA and the amino acid sequences of humanized versions 1.01 and 1.21 are light chain variable regions that are the same and are shown in Figure 16. The heavy chain of cDNA and the amino acid sequences of humanized versions 1.01 and 1.21 are shown in Figure 17A and B.

Example 13: Expression and purification of huDS0 in CHO cells and affinity measurements To determine whether the humanized DS6 versions retained the binding affinity of muDS6 it was necessary to express and purify the antibodies. CHO cells were stably transfected with a chimeric version of DS6 (chDSó) having a human constant region and the variable region of murine, or with huDSóvl.01 or huDSóvl.21. CHODG44 cells (4.32 x 106 cells / plate) were seeded in 15 cm plates in non-selective medium (Alpha MEM + nucleotides (Gibco), supplemented with 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin , and 10% v / v FBS) and placed in a humidified 37 ° C, 5% CO2 incubator. The next day, the cells were transfected with the chDS6 expression plasmid using a modified version of the Qiagen protocol recommended for Polyfect transfection. Non-selective media were aspirated from the cells. The plates were washed with 7 ml of pre-warmed (37 ° C) PBS and re-incubated with 20 ml of non-selective media. Plasmid DNA (11 μg) was diluted in 800 μl of Hybridoma SFM (Gibco). Then, 70 μl of Polyfect (Qiagen) were added to the DNA / SFM mixture. The Polyfect mixture was then vortexed moderately for several seconds and incubated for 10 minutes at room temperature. Non-selective media (2.7 ml) was added to the mixture. This final mix was incubated with the cells on a plate for 24 hours. The mixture / transfection medium was removed from the plates and the cells were trypsinized and counted. Cells were plated on selective media (Alpha MEM-nucleotides, supplemented with 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin, 10% v / v FBS, 1.25 mg / ml G418) in 96 well plates (250 μl / well) in various densities (1800, 600, 200, and 67 cells / well). The cells were incubated for 2-3 weeks, supplementation medium if necessary. Wells were screened for antibody production levels using a quantitative ELISA. An Immulon 2HB 96 well plate was coated with goat anti-human IgG F (ab) 2 antibody (Jackson Immunoresearch; 1 μg / well in 100 μl 50 mM sodium carbonate regulator pH 9.6) and incubated for 01.5 h at room temperature, with oscillation. All subsequent stages were conducted at room temperature. The wells were washed twice with T-TBS (0.1% Tween-20, TBS) and blocked with 200 μl of blocking buffer (1% BSA, T-TBS) for 1 hour. The wells were washed twice with T-TBS. On a separate plate, the standard antibody, EM164 (100 ng / ml), and the culture supernatants were serially diluted (1: 2 or 1: 3) in blocking buffer. These dilutions (100 μl) were transferred to the ELISA plate and incubated for 1 hour. The wells were washed 3 times with T-TBS and incubated with 100 μl goat anti-human IgG Fc-AP (Jackson ImmunoResearch) diluted 1: 3000 in blocking regulator for 45 minutes After 5 washes with T-TBS, the wells were developed using 100 μl of PNPP development reactive (10 mg / ml PNPP (p-Nitrophenyl Phosphate, Disodium Salt; Pierce), 0.1 M diethanolamins pH 10.3 buffer) for 25 minutes. Absorbance at 405 nm was measured in an ELISA plate reader. Absorbance readings (from the culture supernatant) in the linear portion of the standard curve were used to determine antibody levels. The highest production clones, identified by ELISA, were then subcloned, expanded and frozen cell stocks were prepared. For the expression of huDSovl.01 and huDS6vl.21, DG44 CHO cells (Dr. Lawrence Chasin, Columbia University, NY) were cultured in Alpha MEM with ribonucleosides and deoxyribonucleosides (Gibco catalog # 12571, Grand Island, New York). The medium was supplemented with 10% fetal bovine serum (catalog # HyClone SH30071.03, Logan, UT), 1% gentamicin (catalog # Mediatech 30-005-CR, Hemdon, VA), and 2mM L-glutamine (L-glut) (catalog # BioWhittaker 17-605E, Waikersville, MD). This formulation was called CHO Complete Medium. DG44 CHO cells (5x106) were transfected with 50 μg of huDSó DNA plasmid. Before transfection, the cells were removed from the flasks with trypsin (# of Gibco actin 15090-046, Grand Island, NY) and washed twice with non-supplemented Alpha MEM lacking ribonucleosides and deoxyribonucleosides (# Catalog Gibco 12561, Grand Island, NY). This was called Washing Medium. The cells were mixed with plasmid DNA in electrode cells with 0.4 cm of space (catalog # BioRad 1652088, Hercules, PA). They were placed on ice for two minutes and then boosted to 1,000 μF and 260 volts in a BioRad electrophoretic device. After electrophoration, the cells were incubated on ice for two minutes. The cells were then placed in plates in five 24-well plates (catalog # Costar 3524) in Complete Medium CHO and kept in an incubator at 37 ° C with 5% CO2. After 48 hours, the medium was removed from the wells. The wells were rinsed once with Wash Medium and fed with Alpha MEM without ribonucleosides and deoxyribonucleosides (catalog # Gibco 12561, Grand Island, NY) supplemented with 1% gentamicin, 2 mM L-glut, 10% dialysed fetal bovine serum ( catalog # Gibco 26400-044, Grand Island, NY), and 1.25 mg / mL geneticin (G418) (catalog # 1181 1, Grand Island, NY). This complete formulation was called the Selection Medium. The clones were incubated in the Selection Medium during approximately two weeks at which time they were screened for antibody production by Quantitative ELISA. The clones of higher production were then subcloned, expanded and frozen cell stocks were prepared. To produce a sufficient amount of antibody for purification, the cells were expanded on 15 cm plates (~ 1 x 106 cells / plate) with 30 ml of selective medium supplemented with Ultra Low IgG FBS (Gibco) and incubated for 1 week. The culture supernatants were collected in 250 ml conical tubes, rotated in a tabletop centrifuge (2000 rpm, 5 minutes, 4 ° C), and then sterilized-filtered through a 0.2 μm filter apparatus. For purification of DS6, NaOH pellets were added to the culture supernatants filtered to a final pH of 8.0. A column A Hi Trap rProtein (Amersham) was equilibrated with 20-50 column volumes of binding buffer. The supernatant was loaded onto the column using a peristaltic pump. Then, the column was washed with 50 column volumes of binding buffer. The bound antibody was eluted from the column using elution buffer (100 mM acetic acid, 50 mM NaCl, pH 3) in tubes fixed in a fraction collector. The eluted antibody was neutralized using neutralization buffer (2 M K2HPO4) pH 10.0) and dialyzed overnight in PBS. The dialysed antibody was filtered through a syringe filter of 0.2. The absorbance at 280 nm was measured to determine the final protein concentration. The affinity of the purified hulgG was compared to muDS6 by flow cytometry. In the first set of experiments the direct link was measured for a cell line expressing CA6, KB. As shown in Figure 18, muDSß, chDS6, huDSóvI.OI and huDS6 vl .21 show very similar affinities with apparent Kds of 0. 82 nM, 0.69 nM, 0.82 and 0.85 nM, respectively, suggesting that the coating has not altered the CDRs. To confirm that huDS6 versions retain the affinity of muDS, competitive binding experiments were conducted. The advantage of this format is that the same detection system is used for both murine and human antibodies; that is, biotin-muDS6 / streptavidin-DTAF. The results of the competitive binding assay comparing the ability of muDS6, chDS6, huDSóvI.OI and huDS6vl.21 to compete with biotin-DS6 are shown in Figure 19. The apparent EC50 is 1.9 nM, 1.7 nM, 3.0 and 1.9 nM for muDS, chDS6, huDS6 vl .01, and huDS6 vl.21, respectively. These results indicate that the coating of muDS6 produces a humanized DS6 resulting in little reduction in binding affinity.

Example 14: Preparation of the cytotoxic conjugate muDS6-DMI The muDS6 antibody (8 mg / ml) was modified using 8-fold molar excess of N-succinimidyl-4- (2-pyridyldithio) pentanoate (SPP) to introduce dithiopyridyl groups. The reaction was carried out in 95% v / v Regulator A (50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v / v DMA for 2 hours at room temperature. The slightly turgid reaction mixture was filtered by gel through a NAP or Sephadex G25 column (balanced in Regulator A). The degree of modification was determined by measuring the absorbance of the antibody at 280 nm and the DTT liberated 2-mercaptopyridine (Spy) at 280 and 343 nm. The modified muDS6 was then conjugated to 2.5 mg Ab / mL using a 1.7-fold molar excess of N2-deacetyl-IV-2 (3-mercapto-l-oxopropyl) -maitansine (L-DMI) on Spy. The reaction was carried out in Regulator A (97% v / v) with DMA (3% v / v). The reaction was incubated at room temperature overnight and -20 hours. The opaque reaction mixture was centrifuged (1162 xg, 10 min) and the supernatant was filtered by gel through a NAP-25 or S300 column (Tandem 3, 3x 26/10 desalination columns, G25 medium) equilibrated in Regulator B (IX PBS pH 6.5). The pellet was discarded. The conjugate was sterile filtered using a 0.22 μm Millex-GV filter and dialyzed in Regulator B with a Slide-A-Lyzer. The number of bound DMI molecules per molecule of muDS6 was determined by measuring the absorbance at both 252 nm and 280 nm of the filtrate. It was found that the DMI / Ab ratio is 4.36 and the stage product of the conjugated MUDS6 was 55%. The conjugate antibody concentration was 1.32 mg / mL. The purified conjugate was biochemically chatercterized through size exclusion chromatography (SEC) and was found to be 92% monomer. Analysis of DMI in the purified conjugate indicated that 99% was covalently bound to the antibody. In Figure 20, the flow cytometric linkage of the muDS6-DMI conjugate and muDS6 unmodified to Caov-3 cells shows that conjugation of muDSo results in only a slight affinity loss.

Example 15: In vitro cytotoxicity of muDS6-DMI As an uncovered antibody, muDS6 has not shown proliferative or growth inhibitory activity in cell cultures (Figure 21) However, when muDSo is incubated with cells in the presence of a DMI conjugate for heavy and light chain of Goat anti-mouse IgG , muDS6 is very effective in the targeting and delivery of this conjugate to the cell resulting in indirect cytotoxicity (Figure 21). To further test the inherent activity of uncovered muDS6, a complement-dependent cytotoxicity (CDC) assay was conducted using muDSo. HPAC and ZR-75-1 cells (25,000 cells / well) were plated in 96-well plates, in the presence of 5% human or rabbit serum and several dilutions of muDS, in 200 μl of RHBP medium (RPMI-1640). , 0.1% BSA, 2OmM HEPES (pH 7.2-7.4), 100 U / ml penicillin and 100 ug / ml streptomycin). The cells were incubated for 2 hours at 37 ° C. Then Alamar Blue reagent (10% final concentration) (Biosource) was added to the supernatant. The cells were incubated for 5-24 hours before measuring the fluorescence. The murine DS6 had no effect in a complement-dependent cytotoxicity (CDC) assay (Figure 22). This suggests that the therapeutic application of muDS6 would require the conjugation of a toxic effector molecule. The cytotoxicity of the conjugated maitansinoid muDSo antibody was examined using 2 different assay formats in several DS6 positive cell lines. Clonogenic assays were conducted where cells (1000-2500 cells / well) were placed in 6-well plates in 2 ml of diluted conjugate in culture medium. The cells were continuously exposed to the conjugate in various concentrations, usually between 3 x 10"M to 3 x 1?" 9 M, and were incubated in a humidified chamber at 37 ° C, 6% CO2 for 5-9 hours. days. The wells were washed with PBS and the colonies were stained with a solution of 1% w / v crystal violet / 10% v / v formaldehyde / PBS. The unlinked stain was then washed completely from the wells with distilled water, and the plates were allowed to dry. The colonies were counted using a Leica StereoZoom 4 dissecting microscope. Plaque placement efficiency (PE) was calculated as the number of colonies / number of cells in plaques. The surviving fraction was cleaved as PE of treated / PE cells from untreated cells. The IC50 concentration was determined by plotting the surviving fraction of cells against the molar concentration of the conjugate. In a clonogenic trial (Figure 23), muDS6-DMI was effective in removing Caov-3 cells with an estimated IC50 of 800 pM. Negative antigen cells, A375, were only slightly affected by the conjugate at a concentration of 3 x 10'9 M, the highest concentration of the muDS6-DMI tested, demonstrating that the cellular elimination activity of the conjugate is directed specifically towards the cells that express antigen. However, despite the apparent sensitivity to maytansine, many of the other DS6 positive cell lines were not particularly sensitive to the immunoconjugate. All the cervical cell lines (HeLa, KB, and WISH) were sensitive to the conjugate whereas only a select number of the ovarian and breast cell lines showed any cytotoxic affectations. None of the pancreatic cell lines seemed to have been affected. In the MTT assay, cells were seeded in 96-well plates at a density of 1000-5000 cells / well. Cells were plated on serial dilutions of muDS6 or muDS6-DMI immunoconjugate in 200 μl of culture medium. The samples were operated in triplicate. Cells and antibody / conjugate samples were then incubated for 2-7 days, at which time cell viability was determined by means of an MTT assay ([3 (4,5-dimethylthiazol-2-yl) -2,5- diphenyl tetrazolium bromide)].

MTT (50 μg / well) was added to the culture supernatant and allowed to incubate for 3-4 hours at 37 ° C. The medium was removed and the MTT formazan was solubilized in DMSO (175 μl / well). Absorbance was measured at 540-545 nm. In a MTT cell viability assay (Figure 24C), the immunoconjugate was able to effectively remove Caov-3 cells with an estimated IC50 of 1.61 nM. The wells with the highest concentrations of conjugate did not contain viable cells compared to the uncovered antibody which had no effect (Figures 21 and 24). The results of the MTT tests on the other lines Cells were slightly different (Figure 24A, B, and D-1). In many cases, although some cytotoxicity was observed, the conjugate was not able to completely eliminate the cell population (with the exception of WISH cells). The BT-20, OVCAR5, and HPAC cells were particularly resistant: at the highest conjugate concentration (32 nM) in the wells, more than 50% of the cells were still viable.

Example 16: Anti-tumor Activity of In Vivo Conjugate.

To demonstrate the in vivo activity of the muDS6-DMI conjugate, human tumor xenografts were established in SCID mice. A subcutaneous model of the human cervical carcinoma cell line, KB, was developed. The KB cells were cultured in vitro, harvested, and 5 x 1 O6 cells in 100 μL of a serum free medium were injected under the right man of each mouse and allowed to grow for 6 days at an average tumor volume of 144 ± 125 mm3 at which time the drug treatment was started. The mice were given PBS, conjugated at 150 μg / kg DMI, or conjugated at 225 μg / kg DMI (2 mice plor group) intravenously every other day for 5 days. The toxic responses were monitored daily during the treatment. The tumor volumes (Figure 25A) and the corresponding body weights (Figure 25B) were monitored throughout the study. KB tumors treated with PBS control grew quickly with a doubling time of approximately 4 days. In contrast, both groups of mice treated with the conjugate exhibited complete tumor regression at 14 days and at 18 days after the start of treatment for the dose groups of 225 μg / kg and 150 μg / kg, respectively. At the dose of 150 μg / kg the tumor delay was approximately 70 days. Treatment at 225 μg / kg resulted in cures since there was no evidence of tumor recurrence at the end of the study at day 120. As seen in Figure 25B, mice in the 150 μg / kg group showed no loss of weight indicated that the dose was well tolerated. At the highest dose, the mice experienced only a temporary reduction of 3% of body weight. During the course of treatment of 5 days, the mice did not exhibit visible signs of toxicity. Taken together, this study demonstrates that the treatment of muDS6-DMI can cure mice of KB xenograft tumors in a non-toxic dose. The activity of muDS6-DMI was further tested in a panel of subcutaneous xenograft models (see Figure 26). The tumor cell lines used to make xenografts exhibited a range of in vitro maytans sensitivities and CA6 epitope densities (Table 9 below). OVC AR5 and TOV-21G cells are ovarian tumor cell lines; HPAC is a pancreatic tumor cell line; HeLa is a cervical tumor cell line. The OVC AR5 and TOV-21 G cells have low expression of surface CA6; HeLa cells have a level intermediate expression of surface CA6; HPAC cells have a high CA6 density of surface expression. TOV-21G and HPAC cells are sensitive to maytansine; OVC AR5 and HeLa cells are 2-7-fold less sensitive to maytansine.

Table 9 * average maximum relative fluorescence average The 4 cell lines were cultured in vitro, harvested, and 1 x 107 cells in 100 μL of a serum-free medium were injected under the right man of each mouse (6 mice per model) and allowed to grow for 6 days to a volume mean tumor of 57.6 ± 6.7 and 90.2 ± 13.4 mm3 for the test and control groups respectively of OVCAR5, 147.1 ± 29.6 and 176.2 ± 18.9 mm3 for the test and control groups respectively of HPAC, 194.3 ± 37.2 and 201.7 ± 71.7 mm3 for the test and control groups respectively of HeLa, and 96.6 ± 22.8 and 155.6 ± 13.4 mm3 for the test and control groups respectively of TOV-2 IG, at which time the drug treatment was started. For each model, three control mice were treated with two weekly doses of PBS and three test mice were treated with two weekly doses of conjugate (600 μg / kg DMI) intravenously. Toxic responses were monitored daily during treatment and tumor volumes and body weights were monitored throughout the study. The efficiency of the conjugate for the different models is shown graphically in Figures 26A, C, E, and G and the corresponding body weights are plotted in Figures 26B, D, F, and H. The cell lines OVC AR5, TOV -21G, and HPAC form aggressive tumors as can be seen in the PBS controls for each model. The HeLa model had a lag period of approximately 3 weeks before the start of exponential growth. In all the models, treatment with DS6-DM1 conjugate had resulted in a complete regression of the tumor in all mice. For the TOV-21G, HPAC, and HeLa models the mice remained tumor-free at day 61. In the OVC AR5 model there was a recurrence of the tumor at approximately day 45 after tumor inoculation. Therefore, the treatment of muDS6-DMI in this model results in a retraction of tumor growth of approximately 34 days. The growth delay is significant since the OVCAR5 cells are less sensitive to maytansine and have low expression of CA6 epitope. In models where the epitope density CA6 is higher or the model has greater sensitivity to maytansine, the regression of the tumor is more solid. It is important to note that only 2 doses were administered. Clearly the dosing schedule used in this study was not toxic to the mice since no weight loss was observed. It is likely that the cures could be achieved with additional or higher doses of conjugate. Human ovarian cancer is by far an enfeeblement of the peritoneum. OVC AR5 cells grow in a graded manner as an intraperitoneal (IP) model in SCID mice forming tumor nodules and producing ascites in a manner similar to a human disease. To demote activity in an IP model, muDS6-DMI was used to treat mice that have OVCAR5 IP tumors (Figure 27). OVC AR5 cells were developed in vitro, cultured and 1 x 107 cells in 100 μL of serum free medium were injected intraperitoneally. The tumors were allowed to grow for 6 days at which time the treatment was started. The mice were treated weekly for 2 weeks with PBS or DS6-DM1 conjugate at a dose of 600 μg / kg DMI and monitored for weight loss resulting from peritoneal disease. By day 28, the PBS group of mice had lost more than 20% of body weight and were sacrificed. The treated group was sacrificed on day 45 after exceeding a 20% body weight loss. This study shows that muDS6-DMI it is able to delay tumor growth in the aggressive OVCAR5 EP model despite the fact that OVCAR5 cells are less sensitive to maytansine and have little CA6s epitope per cell. Because the dosing schedule did not allow for visible signs of toxicity, it is likely that additional and higher doses could be used to achieve additional delay or cure of the tumor.

Example 17: Synthesis and Characterization of Taxoid Cytotoxic Conjugate DS6-SPP-MM 1-202 muDS6 was modified with the linker IV-sulfosuccinimidyl-4-nitro-2-pyridyl-pentanoate (SSNPP). To 50 mg of muDS Ab in 90% Regulator A, 10 equivalents 10% DMA of SSNPP in DMA were added. The final concentration of Ab was 8 mg / ml. The reaction was stirred for 4 hours at room temperature, then purified by means of G25 chromatography. The extent of antibody modification was measured spectrophotometrically using the absorbance at 280 ran (antibody) and 325 (linker) and found to have 3.82 linkers / antibody. The recovery of the antibody was 43.3 mg giving an 87% yield. The conjugation of muDS6-nitroSPP was conjugated with Taxoid MM 1-202 (1812 P.16). The conjugation was carried out on a scale of 42 mg in 90% Regulator A, 10% DMI. The taxoid was added in 4 aliquots of 0.43 eq / linker (each aliquot) during a period of approximately 20 hours. By this time the reaction had become remarkably murky. After the G25 purification the resulting conjugate, recovered in a yield of approximately 64% had approximately 4.3 taxoids / Ab and remained approximately 1 equivalent of unreacted linker. To quench the unreacted linker, 1 unreacted cysteine / linker equivalent was added to the conjugate with stirring overnight. A definite yellowish hue was noticeable to the addition of cysteine indicating the release of thiopyridine. The reaction solution was then dialyzed in Regulator B / 0.01% Tween 20 followed by additional dialysis only in Regulator B for several days. The final conjugate had 2.86 drugs / antibody. The antibody recovery was 14.7 mg, giving a general yield of 35%. The conjugate was additionally biochemically characterized by SEC and found to have 89% monomer, 10.5% dimer and 0.5% higher molecular weight aggregate. The results of a flow cytometric analysis compared to the binding of muDS6-SPP-MM 1-202 taxoid against the muDS6 antibody in HeLa cells is shown in Figure 28. The indicative results that muDS6 retains the binding activity when it is conjugate for a taxane.

Example 18: In vitro and in vivo activity of humanized DS6 conjugate A conjugate huDSóvl.01 -SPDB-DM4 was constructed. This conjugate is similar to the muDS6-SPP-DMI conjugate described in Example 14 except that the linker / maytansin portion of the conjugate differs in structure around the disulfide bond; the muDS6-SPP-DMI conjugate has a methyl group hindrance at the disulfide carbon on the antibody side of the linker while the SPDB-DM4 conjugate has two methyl group impediments at the disulfide carbon on the maytansine side of the linker. The antibody huDSovl.01 (8 mg / ml) was modified using an 8-fold molar excess of N-succinimidyl-4- (2-pyridyldithio) butanoate (SPDB) in order to introduce dithiopyridyl groups. The reaction was carried out in 95% v / v Regulator A (50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v / v ethanol for 1.5 hour at room temperature. The reaction mixture was filtered by gel through a Sephadex G25 15ml column (balanced in Regulator A). The degree of modification was determined by measuring the absorbance of the antibody at 280 nm and the DTT released 2-mercaptopyridine (Spy) at 280 and 343 nm. The modified DS6 was then conjugated to 1.8 mg Ab / mL using a 1.7-fold molar excess of N2-deacetyl-N-2 (4-methyl-4-mercapto-l-oxopentyl) -maitansine (L-DM4) about Spy. The reactions were carried out in Regulator A (97% v / v) with DMA (3% v / v). The reaction was incubated at room temperature overnight for -20 hours. In contrast to the conjugation of muDS6, the reaction mixture was clear and immediately underwent gel filtration through a NAP 15ml G25 column equilibrated in Citrate buffer (20 mM citrate, 135 mM NaCl, pH 5.5). The conjugate was sterile filtered using a 0.22 μm Millex-GV filter. The number of bound DM4 molecules per DS6 molecule was determined by measuring the absorbance at 252 nm and 280 nm of the filtrate. It was found that the DM4 / Ab ratio is 3.2 and the conjugate DS6 stage yield was 69%. The concentration of conjugated antibody was 1.51 mg / mL. The purified conjugate was characterized biochemically by size exclusion chromatography (SEC) and found to be 92.5% monomer. Analysis of DM4 in the purified conjugate indicated that > 99% was covalently bound to the antibody. In Figure 29 A, the flow cytometric linkage of the huDSov conjugate 1.01-DM4 and unmodified DS6 for KB cells shows that the conjugation of huDSovl.01 does not result essentially in loss of affinity. The linkage was conducted in an essential manner as described for Figure 20 except that the KB cells were used in place of the CaOv-3 cells. The in vitro cytotoxicity of huDS0v1.01 was tested essentially as described in Figure 24G. huDSóvl.01 eliminated the WISH cells with an IC50 of 4.4 x 1? 10 M whereas the non-conjugated huDS? v1.0 did not show cytotoxic activity.

The in vivo activity of huDS6v 1.01-DM4 was tested in the pancreatic HPAC model. The HPAC cells were inoculated on day 0, and immunoconjugate treatments were given on day 13. The PBS control animals were sacrificed once the tumor volumes exceeded 1000 mm3. The conjugate was delivered in a dose of 200 μg / kg or 600 μg / kg DM4, corresponding to an antibody concentration of 15 mg / kg and 45 mg / kg, respectively. The tumor volume (Figure 30A) and the body weight (Figure 30B) of the mice were monitored during the course of the study. huDSóvl.01 -DM4 showed potent antitumor activity at 200 μg / kg DM4 with all the mice that achieve complete regression of the tumor. The control B4-DM4 conjugate that recognizes an antigen not expressed in HPAC xenografts essentially did not have activity at 200 μg / kg. The lack of loss of body weight (Figure 30B) of the mice indicates that the treatment with 200 μg / kg of conjugate is below the maximum tolerated dose. This result demonstrates that a humanized version of DS6 is capable of mediating the targeted delivery of a maitansionoid drug resulting in potent anti-tumor activity.

Claims (20)

1. An 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, characterized in that the heavy chain variable region or fragment thereof has at least 90% sequence identity for an amino acid sequence selected from the group comprising NO SEQ ID: 9, NO SEQ ID: 10, and NO SEQ ID NO: 11: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 9) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: l 1).

2. The antibody or epitope binding fragment thereof according to claim 1, further characterized in that the heavy chain variable region or fragment thereof has at least 95% sequence identity for the amino acid sequence of NO. SEC ID: 9, NO SEC ID: 10, or NO SEC ID: 1 1.

3. The antibody or epitope binding fragment thereof according to claim 1, characterized in that the heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO: 9, NO SEQ ID: 10, or NO SEQ ID: 11. The antibody or epitope binding fragment according to claim 1, further characterized in that the light chain variable region or fragment thereof has at least 90% sequence identity for a sequence of amino acid represented by NO SEC ID: 7 or NO SEC ID: 8: QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSL ASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAGTKLEL KR (SEQ ID NO: 7) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSL ASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAGTKLEL KR (SEQ ID NO: 8). 5. The antibody or epitope binding fragment according to claim 1, further characterized in that the light chain variable region or fragment thereof has at least 95% sequence identity for the amino acid sequence of NO. : 7 or NO SEC ID: 8. The antibody or epitope binding fragment according to claim 1, further characterized in that the light chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. 7. The antibody or epitope binding fragment according to claim 1, further characterized in that the heavy chain variable region or fragment thereof has the sequence of amino acid of NO SEC ID: 9, NO SEC ID: 10, or NO SEC ID: 11, and wherein the light chain variable region or fragment thereof has the amino acid sequence of NO SEC ID: 7 or NO ID SEC: 8. A polynucleotide encoding an antibody or an epitope-binding fragment thereof according to claim 1. 9. A polynucleotide encoding an antibody or an epitope-binding fragment thereof in accordance with claim 1.

4. A polynucleotide encoding an antibody or an epitope-binding fragment thereof according to claim 7. 11. A polynucleotide that encodes a heavy chain of an antibody or an epitope-binding fragment thereof. according to claim 1. 12. An expression vector comprising the polynucleotide according to claim 8. 13. An expression vector comprising the polynucleotide according to claim 9. 14. An expression vector comprising the polynucleotide according to claim 10. 1

5. A receptor cell comprising an expression vector according to claim 12. 1

6. A cell receptor comprising an expression vector according to claim 13. 1

7. A receptor cell comprising an expression vector according to claim 14. 1

8. A method for preparing an antibody or an epitope-binding fragment thereof. comprises culturing the receptor cell according to claim 15 under conditions that promote the expression of said antibody or an epitope-binding fragment thereof and the recovery of the polypeptide from the cell culture, characterized in that the antibody or an epitope-binding fragment thereof comprises at least one heavy chain variable region or fragment thereof and at least one light chain variable region or fragment thereof, wherein the variable region The heavy chain or fragment thereof has at least 90% sequence identity for an amino acid sequence selected from the group consisting of NO ID SEC: 9, SEQ ED NO: 10, and NO SEC ID. eleven: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 9) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: l 1). A method for preparing an antibody or an epitope-binding fragment thereof comprising culturing the recipient cell according to claim 16 under conditions that promote the expression of the antibody or an epitope-binding fragment thereof and the recovery of the polypeptide from the cell culture, characterized in that the heavy chain variable region or fragment thereof has at least 90% sequence identity for an amino acid sequence selected from the group consisting of NO SEC ID: 9, NO SEC ID: 10, and NO SEC ID: 11: QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 9) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIG YIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 1), and wherein the light chain variable region or fragment thereof has at least 90% sequence identity for an amino acid sequence represented by NO SEC ID: 7 or NO SEC ID : 8: QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSL ASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAGTKLEL KR (SEQ ID NO: 7) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSL ASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAGTKLEL KR (SEQ ID NO: 8). 20. A method for preparing an antibody or an epitope-binding fragment thereof comprising culturing the recipient cell according to claim 17 under conditions that promote the expression of said antibody or an epitope-binding fragment thereof and recovery. of the polypeptide from the cell culture, characterized in that the heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO: 9, NO SEQ ID: 10, or NO SEQ ID: 11, and wherein the The light chain variable region or fragment thereof has the amino acid sequence of NO SEC ID: 7 or NO SEQ ID: 8.