JP2012161321A - Ca6 antigen-specific cytotoxic conjugate, and method using the conjugate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new cytotoxin conjugate containing a cell binding agent containing antibody recognizing a molecule/receptor expressed on the surface of cancerous cells, and a cytotoxin agent targetting a molecule/receptor expressing on the surface of cancerous cells.SOLUTION: A monoclonal antibody, and epitope-binding fragments thereof recognize and binds the CA6 glycotope. There are provided humanized or resurfaced versions of DS6, an anti-CA6 murine monoclonal antibody, and epitope-binding fragments thereof.

Description

FIELD OF THE INVENTION [01] The present invention relates to murine anti-CA6 glycotope monoclonal antibodies, and their humanized forms or their resurfaced forms. The present invention also relates to epitope-binding fragments of anti-CA6 glycotope monoclonal antibodies, as well as humanized or surface reconstituted epitope-binding fragments of anti-CA6 glycotope monoclonal antibodies.

  [02] The present invention relates to a cytotoxic conjugate comprising a cell binding agent and a cytotoxic agent, a therapeutic composition comprising the conjugate, a method for using the conjugate in inhibiting cell proliferation and treating a disease. As well as a kit comprising said cytotoxic conjugate. In particular, the cell binding agent is a monoclonal antibody or epitope binding fragment thereof, or a humanized or surface reconstituted form thereof that recognizes and binds to the CA6 glycotope.

BACKGROUND OF THE INVENTION [03] Numerous attempts have been made to develop anticancer therapeutics that specifically destroy target cancer cells without harming surrounding non-cancerous cells and tissues. Such therapeutic agents have the potential to greatly improve the treatment of cancer in human patients.

  [04] One promising approach was to link cell binding agents, such as monoclonal antibodies, with cytotoxic agents (Sela et al., In Immunoconjugates 189-216 (supervised by C. Vogel 1987); Gose et al. , Targeted Drugs, 1-22 (supervised by E. Goldberg, 1983); Diener et al., In Antibiotic-delivered delivery systems (supervised by J. Rodwell, 1988); Pietersz, et al., D. 1988); Bumol et al., In the Antibody-mediated delivery systems. 55-79 (supervised by J. Rodwell 1988) Depending on the choice of cell binding agent, only certain types of cancerous cells are recognized based on the expression profile of the molecules expressed on the surface of such cells, and It is also possible to design these cytotoxic conjugates to bind only to these types.

[05] Cytotoxic agents such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil linked to various murine monoclonal antibodies have been used in these cytotoxic conjugates. ing. In some cases, serum albumin (Garnett et al., 46 Cancer Res. 2407-2412 (1986); Ohkawa et al. 23 Cancer Immunol. Immunoother. 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); , 85 Proc.
l. Acad. Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al., 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al. 27 J. Med. Chem. 1602-1607 (1984); 52 Br. J. Cancer 111-116 (1985)), the drug molecule was linked to the antibody molecule.

[06] Examples of one specific conjugate that has been shown to be somewhat promising include the C242 antibody directed against CanAg, an antigen expressed on colorectal and pancreatic tumors, and the maytansine derivative DM1 (Liu et al., Proc Natl Acad Sci USA, 93: 8618-8623 (1996)). In vitro evaluation of this conjugate shows that its binding affinity towards CanAg expressed on the cell surface is high, the apparent K _d value is 3 × 10 ⁻¹¹ M, and its cytotoxicity against CanAg positive cells is high, IC ₅₀ was shown to be 6 × 10 ⁻¹¹ M. This cytotoxicity is antigen dependent because it was blocked by excess unconjugated antibody and because antigen negative cells were more than 100 times less sensitive to the conjugate. Other examples of antibody-DM1 conjugates with high affinity towards their respective target cells and high antigen-selective cytotoxicity include humanized versions of antibodies against human CD56, huN901; antibodies against human CD33 Humanized form, huMy9-6; humanized form of antibody to CanAg Mucl epitope, huC242; deimmunized antibody to PSMA, huJ591; humanized antibody to Her2 / neu, trastuzumab; and humanized antibody to CD44v6, bibuzumab Is included.

  [07] In continuing to improve the methods used to treat cancer patients, it may be important to develop additional cytotoxic conjugates that specifically recognize certain types of cancerous cells.

  [08] To this end, the present invention relates to the development of antibodies that recognize and bind to molecules / receptors expressed on the surface of cancerous cells, and cell-binding agents such as antibodies, and It relates to the development of novel cytotoxic conjugates comprising cytotoxic agents that specifically target molecules / receptors expressed on the surface of cancerous cells.

  [09] More specifically, the present invention recognizes the novel CA6 sialoglycotope on the Mucl mucin receptor expressed by cancerous cells, and recognizes the novel CA6 sialoglycotope of Mucl mucin, and In the context of cytotoxic agents, it relates to the provision of antibodies, preferably humanized antibodies, that can be used to inhibit the growth of cells expressing the CA6 glycotope.

SUMMARY OF THE INVENTION [10] The present invention includes antibodies or epitope-binding fragments thereof that specifically recognize and bind to the novel CA6 sialoglycotope of the Mucl mucin receptor. In another aspect, the invention includes a humanized antibody or epitope-binding fragment thereof that recognizes a novel CA6 sialoglycotope (“CA6 glycotope”) of the Mucl mucin receptor.

[11] In a preferred embodiment, the present invention includes a murine anti-CA6 monoclonal antibody DS6 (“DS6 antibody”), and the surface of the human antibody or epitope-binding fragment thereof, wherein the residues exposed on the surface are known human antibody surfaces. To more closely resemble the surface reconstituted or humanized form of the DS6 antibody, substituted in both the light and heavy chains. The humanized antibodies and epitope-binding fragments thereof of the present invention are improved in that they are much less immunogenic (or completely non-immunogenic) than the fully murine form in the administered human subject. It has the characteristics. Thus, the humanized DS6 antibodies and epitope-binding fragments thereof of the present invention are not immunogenic in humans, while specifically recognizing a novel sialoglycotope on the Mucl mucin receptor, i. Specific cytotoxicity directed against cells expressing the antigen by conjugating the humanized antibody and its epitope-binding fragment to a drug such as maytansinoid and targeting the drug to the Muc1 CA6 sialoglycotope Prodrugs having sex may be formed. Accordingly, cytotoxic conjugates comprising such antibodies and small highly toxic agents (eg, maytansinoids, taxanes, and CC-1065 analogs) can be used as therapeutic agents for the treatment of tumors such as breast and ovarian tumors. It may be used as

  [12] The humanized form of the DS6 antibody of the present invention comprises the amino acid sequences of both the light and heavy chain variable regions, the DNA sequences of the light and heavy chain variable region genes, the CDRs, and the surface amino acids. , As well as the disclosure of means for its expression in recombinant form, is fully characterized herein.

  [13] In one embodiment, the amino acid sequence represented by SEQ ID NOs: 1-3:

An amino acid sequence having a heavy chain comprising a CDR having and represented by SEQ ID NOs: 4-6:

A humanized DS6 antibody or epitope-binding fragment thereof having a light chain comprising a CDR having
[14] Amino acid sequence represented by SEQ ID NO: 7 or 8:

Also provided are humanized DS6 antibodies and epitope binding fragments thereof having light chain variable regions having amino acid sequences that share at least 90% sequence identity.
[15] Similarly, the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11:

And humanized DS6 antibodies and epitope binding fragments having heavy chain variable regions with amino acid sequences sharing at least 90% sequence identity.
[16] In another embodiment, the amino acid sequence corresponding to SEQ ID NO: 8

Humanized DS6 antibodies and epitope-binding fragments thereof having humanized or surface-restructured light chain variable regions having
[17] Similarly, amino acid sequences corresponding to SEQ ID NO: 10 and SEQ ID NO: 11, respectively:

Humanized DS6 antibodies and epitope-binding fragments thereof having humanized or surface-reorganized heavy chain variable regions having
[18] The humanized DS6 antibody and epitope-binding fragments thereof of the present invention also correspond to murine surface framework residues found within 5 angstroms of the CDRs that require changes to human residues, Table 1. Substitutions in the light and / or heavy chain amino acid residues at one or more positions as defined by the asterisk residues can also be included. For example, the first amino acid residue Q in the murine sequence (SEQ ID NO: 7) has been replaced by E (SEQ ID NO: 8) to humanize the antibody. However, since this residue is close to the CDR, a back mutation to murine residue Q may be necessary to maintain antibody affinity.
Table 1

[19] This is further illustrated in Table 2, which shows the muDS6 variable region surface residues in parallel with the three most homologous human variable region surface residues. The amino acid residues in Table 1 correspond to the underlined amino acid residues in Table 2.
Table 2

  [20] The present invention further provides a cytotoxic conjugate comprising (1) a cell binding agent that recognizes and binds to a CA6 glycotope, and (2) a cytotoxic agent. In the cytotoxic conjugate, the cell binding agent has a high affinity for the CA6 glycotope and the cytotoxic agent is CA6, such that the cytotoxic conjugate of the present invention forms an effective killing agent. Has a high degree of cytotoxicity against cells expressing glycotopes.

  [21] In a preferred embodiment, the cell binding agent is an anti-CA6 antibody or epitope-binding fragment thereof, more preferably a humanized anti-CA6 antibody or epitope-binding fragment thereof, wherein the cytotoxic agent is directly Alternatively, it is covalently linked to the antibody or epitope-binding fragment thereof via a cleavable or non-cleavable linker. In a more preferred embodiment, the cell binding agent is a humanized DS6 antibody or epitope binding fragment thereof and the cytotoxic agent is taxol, maytansinoid, CC-1065 or a CC-1065 analog.

[22] In a preferred embodiment of the invention, the cell binding agent is a humanized anti-CA6 antibody and the cytotoxic agent is a cytotoxic agent such as maytansinoid or taxane.
[23] More preferably, the cell binding agent is a humanized anti-CA6 antibody DS6 and the cytotoxic agent is a maytansine compound such as DM1 or DM4.

  [24] The present invention also includes a method for inhibiting the growth of cells expressing a CA6 glycotope. In preferred embodiments, the method for inhibiting the growth of cells expressing the CA6 glycotope occurs in vivo and results in cell death, although in vitro and ex vivo applications are also included.

[25] The present invention also provides a therapeutic composition comprising a cytotoxic conjugate and a pharmaceutically acceptable carrier or excipient.
[26] The present invention further includes a method of treating a subject having cancer using the therapeutic composition. In preferred embodiments, the cytotoxic conjugate comprises an anti-CA6 antibody and a cytotoxic agent. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized DS6 antibody-DM1 conjugate, a humanized DS6 antibody-DM4 or a humanized DS6 antibody-taxane conjugate, and the conjugate is pharmaceutically acceptable. It is administered with a possible carrier or excipient.

[27] The present invention also includes a kit comprising an anti-CA6 antibody-cytotoxic agent conjugate and instructions for use. In a preferred embodiment, the anti-CA6 antibody is a humanized DS6 antibody, the cytotoxic agent is a maytansine compound such as DM1 or DM4, or a taxane, and the instructions include conjugates in the treatment of a subject with cancer. It is for use. The kit also includes the components necessary to prepare the pharmaceutically acceptable formulation, such as a diluent if the conjugate is lyophilized or concentrated, and the components necessary to administer the formulation. It may be included.

  [28] The present invention also includes derivatives of antibodies that specifically bind to and recognize the CA6 glycotope. In preferred embodiments, antibody derivatives are prepared by reconstituting the surface of an antibody that binds to a CA6 glycotope or by humanizing the antibody, wherein the derivative has reduced immunogenicity to the host.

  [29] The present invention further provides a humanized antibody or fragment thereof that is further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radiolabel, phosphor, chromophore, contrast agent or metal ion.

  [30] A diagnostic method of administering the labeled humanized antibody or epitope-binding fragment thereof to a subject suspected of having cancer and measuring or monitoring the distribution of the label in the subject also comprises provide.

  [31] The present invention also provides a method for treating a subject having cancer by administering the humanized antibody conjugate of the present invention alone or in combination with other cytotoxic agents or therapeutic agents. Also provide. The cancer is CA6 expressed, eg, breast cancer, colon cancer, ovarian cancer, endometrial cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, sarcoma or carcinoma, or CA6 glycotope May be one or more of other cancers that are predominantly expressed and have not yet been determined.

  [32] Unless otherwise noted, all references and patents cited herein are hereby incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION [63] The present invention provides, among other features, anti-CA6 monoclonal antibodies, anti-CA6 humanized antibodies, and anti-CA6 antibody fragments. Each of the antibodies and antibody fragments of the present invention are designed to specifically recognize and bind to the CA6 glycotope on the cell surface. CA6 is found in many human tumors: 95% of serous ovarian cancer, 50% of endometrial ovarian cancer, 50% of cervical neoplasms, 69% of endometrial neoplasms, and 80% of vulvar neoplasms. It is known to be expressed by 60% of breast cancer, 67% of pancreatic tumors, and 48% of urothelial tumors, but is rarely expressed by normal human tissues.

  [64] Kearse et al., Int. J. et al. Cancer 88 (6): 866-872 (2000) reports that when hybridoma supernatants are used to characterize the CA6 epitope, the protein in which the CA6 epitope is found is expressed as an 80 kDa protein having an N-linked carbohydrate containing the CA6 epitope. I misunderstood. Subsequently, the inventors used purified DS6 to demonstrate that CA6 epitopes are found on O-linked carbohydrates that are larger than the 250 kDa non-disulfide-linked glycoprotein. Furthermore, the glycoprotein was identified as mucin, Muc1. Because different Muc1 alleles have diverse numbers of tandem repeats in the variable number tandem repeat (VNTR) domain, cells often express two distinct Muc1 proteins of different sizes (Taylor-Papadimitriou, Biochim. Biophys. Acta 1455 (2-3): 301-13 (1999) Since the number of repeats in the VNTR domain is different and the glycosylation is also different, the molecular weight of Muc1 varies between cell lines.

  [65] The sensitivity of CA immunoreactivity to periodate indicates that CA6 is a carbohydrate epitope “glycotope”. The further sensitivity of CA6 immunoreactivity to treatment with neuraminidase from Vibrio cholerae indicates that the CA6 epitope is a sialic acid-dependent glycotope and therefore a “sialoglycotope”.

  [66] Details of CA6 characterization can be found in Example 2 (see below). Further details regarding CA6 can be found in WO 02/16401; Wennenberg et al., Am. J. et al. Pathol. 143 (4): 1050-1054 (1993); Smith et al., Human Antibodies 9: 61-65 (1999); Kearse et al., Int. J. et al. Cancer 88 (6): 866-872 (2000); Smith et al., Int. J. et al. Gynecol. Pathol. 20 (3): 260-6 (2001); and Smith et al., Appl. Immunohistochem. Mol. Morphor. 10 (2): 152-8 (2002).

  [67] The present invention also includes cytotoxic conjugates comprising two major components. The first component is a cell binding agent that recognizes and binds to the CA6 glycotope. The cell binding agent should recognize the CA6 sialoglycotope on Muc1 with a high degree of specificity so that the cytotoxic conjugate recognizes only and binds to the intended cell. . A high degree of specificity will allow the conjugate to act in a targeted manner with few side effects resulting from non-specific binding.

  [68] In another embodiment, allowing the conjugate to be internalized by a period of time and / or sufficient to allow the cytotoxic drug portion of the conjugate to act on the cell. The cell binding agent of the invention will also recognize the CA6 glycoepitope with a high degree of affinity so that the conjugate is in contact with the target cell for a sufficient period of time to do so.

  [69] In a preferred embodiment, the cytotoxic conjugate comprises an anti-CA6 antibody, more preferably a murine DS6 anti-CA6 monoclonal antibody, as a cell binding agent. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized DS6 antibody or epitope binding fragment thereof. The DS6 antibody is capable of recognizing CA6 with a high degree of specificity and directs cytotoxic agents to abnormal cells or tissues such as cancer cells in a targeting fashion.

  [70] The second component of the cytotoxic conjugate of the present invention is a cytotoxic agent. In preferred embodiments, the cytotoxic agent is taxol, a maytansinoid such as DM1 or DM4, CC-1065 or a CC-1065 analog. In a preferred embodiment, the cell binding agent of the invention is covalently attached to the cytotoxic agent directly or via a cleavable or non-cleavable linker.

[71] Cell binding agents, cytotoxic agents, and linkers are discussed in more detail below.
Cell-binding agents [72] The effectiveness of the compounds of the present invention as therapeutic agents depends on careful selection of appropriate cell-binding agents. Cell binding agents may be of any kind now known or become known, and such agents include peptides and non-peptides. A cell binding agent may be any compound capable of binding to cells in either a specific or non-specific manner. In general, these may be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (such as transferrin), or any other cell binding molecule or substance.

[73] More specific examples of cell binding agents that can be used include:
(A) a polyclonal antibody;
(B) a monoclonal antibody;
(C) Fab, Fab ′, and antibody fragments such as F (ab ′) ₂ , Fv (Parham, J. Immunol. 131: 2895-2902 (1983); Spring et al. J. Immunol. 113: 470-478 (1974) Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244 (1960));
(D) interferons (eg alpha, beta, gamma);
(E) lymphokines such as IL-2, IL-3, IL-4, IL-6;
(F) Hormones such as insulin, TRH (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgens and estrogens;
(G) Growth factors and colony stimulating factors such as EGF, TGF-alpha, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984));
(H) transferrin (O'Keefe et al. J. Biol. Chem. 260: 932-937 (1985)); and (i) vitamins such as folic acid.

Antibody [74] The selection of the appropriate cell binding agent is a matter of choice depending on the particular cell population to be targeted, but in general, appropriate ones are available or can be prepared. If present, antibodies, more preferably monoclonal antibodies are preferred.

  [75] Monoclonal antibody technology enables the production of highly specific cell binding agents in the form of specific monoclonal antibodies. Particularly well known in the art are mice, rats, hamsters, or any other mammal that are intact target cells, antigens isolated from target cells, complete viruses, attenuated A technique for generating monoclonal antibodies produced by immunization with an antigen of interest, such as a complete virus and a viral protein such as a viral coat protein. Sensitized human cells can also be used. Another method of generating monoclonal antibodies is the use of phage libraries of scFvs (single chain variable regions), particularly human scFvs (see, eg, Griffiths et al., US Pat. Nos. 5,885,793 and 5, 969,108; McCafferty et al., WO 92/01047; Liming et al., WO 99/06687).

[76] A typical antibody is composed of two identical heavy chains and two identical light chains linked by disulfide bonds. A variable region is a portion of an antibody heavy and light chain that varies in sequence within an antibody and cooperates in the binding and specificity of each particular antibody to an antigen. Variability is usually not evenly distributed throughout the antibody variable region. Typically, they are concentrated in three segments of variable regions called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable regions. The higher conserved portion of the variable region is called the framework region. The variable region of the heavy and light chains contains four framework regions, most of which adopt a beta-sheet configuration, each framework region being linked by three CDRs, which are in a beta-sheet structure Are formed, and in some cases, form part of the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen binding site of the antibody (EA Kabat et al. Sequences of Proteins of Immunological Interest, 5th edition, 1991, NIH).

[77] The constant region is part of the heavy chain. Although not directly involved in antibody binding to antigen, it exhibits a variety of effector functions such as antibody involvement in antibody-dependent cytotoxicity.
[78] Monoclonal antibodies suitable for use in the present invention include murine DS6 monoclonal antibody (US Pat. No. 6,596,503; ATCC Deposit Number PTA-4449).

Humanized or surface reconstituted DS antibody [79] Preferably, a humanized anti-CA6 antibody is used as the cell binding agent of the present invention. A preferred embodiment of such a humanized antibody is a humanized DS6 antibody or an epitope-binding fragment thereof.

[80] The goal of humanization is to reduce the immunogenicity of heterologous antibodies, such as murine antibodies, for introduction into humans while maintaining the full antigen binding affinity and specificity of the antibody.
[81] Humanized antibodies may be produced using several techniques such as surface reconstruction and CDR grafting. Herein, surface reconstruction techniques use a combination of molecular modeling, statistical analysis and mutagenesis to modify the non-CDR surface of the antibody variable region to resemble the surface of a known antibody in the target host.

  [82] Strategies and methods for antibody surface reconstitution, as well as other methods for reducing the immunogenicity of antibodies in different hosts, are incorporated herein in their entirety. , 639, 641 (Pedersen et al.). Briefly, in a preferred method, (1) a position alignment of a pool of antibody heavy and light chain variable regions is generated, resulting in a set of heavy and light chain variable region framework surface exposed positions, where all The variable region alignment positions of at least about 98% are identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) Identify the set of heavy and light chain variable region framework surface exposed amino acid residues that are most closely identical to the set of rodent surface exposed amino acid residues; (4) Complementation of rodent antibodies Heavy and light chain variable region framework as defined in step (2), except for amino acid residues within 5 cm of any atom of any residue of the sex determining region Replacing the set of surface exposed amino acid residues with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3); and (5) a humanized rodent with binding specificity Produces antibodies.

[83] CDR grafting (EP 0 239 400; WO 91/09967; US Pat. Nos. 5,530,101; and 5,585,089), surface finishing or surface reconstruction (EP 0 592 106 EP 0 519 596;
Padlan E.M. A. , 1991, Molecular Immunology 28 (4/5): 489-498; Studnika G., et al. M.M. 1994, Protein Engineering 7 (6): 805-814; Roguska M. et al. A. Et al., 1994, PNAS 91: 969-973), and chain shuffling (US Pat. No. 5,565,332) may be used to humanize antibodies. Human antibodies may be made by a variety of methods known in the art, including phage display methods. U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Patent Application Publications WO 98/46645, WO 98/50433, See also WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, which are incorporated herein in their entirety.

  [84] In a preferred embodiment, the present invention provides a humanized antibody or fragment thereof that recognizes a novel sialoglycotope (CA6 glycotope) on the Mucl mucin. In another embodiment, the humanized antibody or epitope binding fragment thereof has the additional ability to inhibit the growth of cells expressing the CA6 glycotope.

  [85] In a more preferred embodiment, a surface reconstituted or humanized version of the DS6 antibody is provided, wherein the surface exposed residues of the antibody or fragment thereof are light so that they more closely resemble a known human antibody surface. Substituted in both the chain and heavy chain. The humanized DS6 antibody or epitope-binding fragment thereof of the present invention has improved properties. For example, the humanized DS6 antibody or epitope-binding fragment thereof specifically recognizes a novel sialoglycotope (CA6 glycotope) on the Mucl mucin. More preferably, the humanized DS6 antibody or epitope-binding fragment thereof has the additional ability to inhibit the growth of cells expressing the CD6 glycotope. Specific cytotoxicity is directed towards antigen-expressing cells by conjugating a humanized antibody or epitope-binding fragment thereof to a drug such as maytansinoid and targeting the drug to a novel Mucl sialoglycotope, CA6. Prodrugs may be formed. Cytotoxic conjugates comprising such antibodies, and small and highly toxic agents (eg maytansinoids, taxanes, and CC-1065 analogs), therapeutic agents for the treatment of tumors such as breast and ovarian tumors It may be used as

  [86] The humanized form of the DS6 antibody also includes the respective amino acid sequences of both the light and heavy chain variable regions, the DNA sequences of the light and heavy chain variable region genes, the CDRs, the surface amino acids, and The disclosure of the means for its expression in recombinant form is also fully characterized herein.

  [87] In one embodiment, the amino acid sequence represented by SEQ ID NOs: 1-3:

A humanized antibody or epitope-binding fragment thereof having a heavy chain comprising a CDR having
[88] When the heavy chain CDRs are determined by AbM modeling software, the heavy chain CDRs are SEQ ID NOs: 20-22:

Indicated by.
[89] In the same embodiment, the humanized antibody or epitope-binding fragment thereof has the amino acid sequence represented by SEQ ID NOs: 4-6:

Having a light chain comprising a CDR having
[90] Amino acid sequence represented by SEQ ID NO: 7 or 8:

Also provided are humanized antibodies and epitope-binding fragments thereof having light chain variable regions having amino acid sequences that share at least 90% sequence identity.
[91] Similarly, the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11:

And humanized antibodies and epitope-binding fragments thereof having heavy chain variable regions with amino acid sequences that share at least 90% sequence identity.
[92] In another embodiment, the amino acid sequence corresponding to SEQ ID NO: 8

Humanized antibodies and epitope-binding fragments thereof having a humanized or surface reshaped light chain variable region having
[93] Similarly, an amino acid sequence corresponding to SEQ ID NO: 10 or SEQ ID NO: 11:

Humanized antibodies and epitope-binding fragments thereof having a humanized or surface rearranged heavy chain variable region having
[94] The humanized antibody of the present invention and its epitope-binding fragment also have human surface amino acid residues adjacent to the CDR marked with an asterisk in Table 1 to maintain the binding affinity and specificity of muDS6. Light chain and / or heavy chain variable region types may also be included, which are substituted by the corresponding muDS6 surface residue at one or more positions defined by the appended residues (Kabat numbering).
Table 1

[95] Disclosed herein are the primary amino acid and DNA sequences of the DS6 antibody light and heavy chains and humanized forms thereof. However, the scope of the present invention is not limited to antibodies and fragments comprising these sequences. Instead, all antibodies and fragments that specifically bind to CA6 as a unique tumor-specific glycotope on the Mucl 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, such antibodies further substantially avoid agonist activity. Thus, the antibodies and antibody fragments of the present invention may differ from the DS6 antibody or humanized derivative thereof in the backbone, CDR, and / or light and heavy chain amino acid sequences and still belong within the scope of the present invention. Is possible.

  [96] Modeling identifies the CDRs of the DS6 antibody and predicts its molecular structure. Again, CDRs are important for epitope recognition but are not essential to the antibodies and fragments of the invention. Thus, antibodies and fragments with improved properties, for example produced by affinity maturation of the antibodies of the invention are provided.

  [97] The murine light chain IgVκ ap4 germline gene and heavy chain IgVh J558.41 germline gene, which appears to be derived from DS6, are shown in FIG. 11 aligned with the sequence of the DS6 antibody. This comparison identifies possible somatic mutations in the DS6 antibody, including some in the CDRs.

  [98] The sequences of the heavy and light chain variable regions of the DS6 antibody, as well as the sequence of the CDRs of the DS6 antibody, are not previously known and are shown in FIGS. 9A and 9B. Such information may be used to generate humanized forms of DS6 antibodies.

Antibody Fragments [99] Antibodies of the present invention include both full length antibodies as discussed above as well as epitope binding fragments. As used herein, “antibody fragment” includes any portion of an antibody that retains the ability to bind to an epitope recognized by a full-length antibody, and these are 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 Fv (scFv), single chain antibody, disulfide linked Fv (dsFv), and Fragments that include either the _VL or _VH regions are included. Epitope binding fragments, including single chain antibodies, can be used alone or in combination with all or part of the following: hinge region, C _H 1, C _H 2 and C _H 3 domains. ) May be included.

[100] Such fragments may contain one or both Fab fragments or F (ab ′) ₂ fragments. Preferably, antibody fragments contain all 6 CDRs of a complete antibody, but fragments containing fewer than all such regions, eg, 3, 4 or 5 CDRs, will also function. Further, the fragment may be a member of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and subclasses thereof, or a combination thereof.

[101] Fab and F (ab ′) ₂ fragments may be produced by proteolytic cleavage using enzymes such as papain (Fab fragments) or pepsin (F (ab ′) ₂ fragments).

[102] The single chain FV (scFv) fragment comprises an epitope comprising at least one fragment of an antibody heavy chain variable region (V _H ) linked to at least one fragment of an antibody light chain variable region (V _L ) It is a binding fragment. The linker ensures proper three-dimensional folding of the (V _L ) and (V _H ) regions when linked to maintain the target molecule binding specificity of the complete antibody from which the single chain antibody fragment is derived. It may be a short, flexible peptide selected as such. Carboxyl terminus of _{(V L)} or _{(V H)} sequences, by a linker, complementary _{(V L)} or _{(V H)} may be covalently bound to the amino terminus of the sequence.

  [103] Single chain antibody fragments of the invention have at least one of the variable or complementarity determining regions (CDRs) of a complete antibody as described herein, but contain some or all of the constant domains of such antibodies. Contains the missing amino acid sequence. These constant domains are not required for antigen binding but constitute a major part of the structure of a complete antibody. Thus, single chain antibody fragments may overcome some of the problems associated with the use of antibodies that contain some or all of the constant domains. For example, single chain antibody fragments tend to be free of unwanted interactions between biological molecules and heavy chain constant regions, or other undesirable biological activities. Furthermore, single chain antibody fragments are much smaller than full antibodies and thus have higher capillary permeability than full antibodies, and single chain antibody fragments are more efficiently localized to the target antigen binding site, And it can be possible to combine. It is also possible to produce antibody fragments on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, because single chain antibody fragments are relatively small in size, they are less likely to elicit an immune response in the recipient than the complete antibody.

  [104] Single chain antibody fragments may be generated by molecular cloning, antibody phage display libraries or similar techniques well known to those skilled in the art. These proteins may be produced in eukaryotic cells or prokaryotic cells including bacteria. In addition, the epitope-binding fragments of the present invention may be generated using various phage display methods known in the art. In the phage display method, a functional antibody domain is displayed on the surface of a phage particle carrying a polynucleotide sequence encoding the antibody domain. In particular, such phage may be utilized to display epitope binding domains expressed from a repertoire or combinatorial antibody library (eg, human or murine). Antigens may be used to select or identify phage that express an epitope binding domain that binds to the antigen of interest, eg, using labeled antigen bound or captured to a solid surface or bead. Phages used in these methods are fd and M13 binding domains expressed from phage containing Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII protein. Typically filamentous phage.

[105] Examples of phage display methods that can be used to generate the epitope-binding fragments of the invention are each described in Brinkman et al., 1995, J. Mol. Immunol. Methods 182: 41-50; Ames et al., 1995, J. MoI. Immunol. Methods 184: 177-186; Kettleborough et al., 1994, Eur. J. et al. Immunol.
24: 952-958; Persic et al., 1997, Gene 187: 9-18; Burton et al., 1994, Advances in Immunology.
PCT Application No. PCT / GB91 / 01134; PCT Publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95 And U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; No. 5,821,047; No. 5,571,698; No. 5,427,908; No. 5,516,637; No. 5,780,225; No. 5,658,727 No .; those disclosed in US Pat. Nos. 5,733,743 and 5,969,108.

[106] After phage selection, the phage region encoding the fragment is isolated and used, for example, using recombinant DNA techniques as described in detail below, in mammalian cells, insect cells, plant cells, yeast And epitope-binding fragments may be generated through expression in a selected host, including bacteria. For example, disclosed 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. Techniques that recombinantly produce Fab, Fab ′, and F (ab ′) ₂ fragments using methods known in the art, such as those described, may also be used; The entirety is hereby incorporated by reference. Examples of techniques that can be used to produce single chain Fv and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology 203: 46-. 88; Shu et al., 1993, PNAS 90: 7995-7999; Skerra et al., 1988, Science 240: 1038-1040.

Functional equivalents [107] Also included within the scope of the invention are functional equivalents of anti-CA6 and humanized anti-CA6 antibodies. The term “functional equivalent” includes, for example, antibodies with homologous sequences, chimeric antibodies, artificial antibodies and modified antibodies, each functional equivalent being defined by its ability to bind to CA6. One skilled in the art will understand that there is an overlap between the group of molecules referred to as “antibody fragments” and the group referred to as “functional equivalents”. Methods for producing functional equivalents are described, for example, in PCT application WO 93/21319, European patent application 239,400; PCT application WO 89/09622, each of which is incorporated herein in its entirety. 338,745; and European Patent Application EP 332,424.

  [108] An antibody having a homologous sequence is an antibody having an amino acid sequence having sequence homology with the amino acid sequences of the anti-CA6 antibody and humanized anti-CA6 antibody of the present invention. Preferably, the homology is homology with the variable region amino acid sequences of the anti-CA6 and humanized anti-CA6 antibodies of the invention. “Sequence homology” as used herein when applied to amino acid sequences is described, for example, in Pearson and Lipman, Proc. Natl. Acad. Sci. At least about 90%, 91%, 92%, 93%, or 94% sequence homology, and more preferably at least about 95, as determined by the FASTA search method according to USA 85, 2444-2448 (1988). Defined as sequences with%, 96%, 97%, 98%, or 99% sequence homology.

  [109] As used herein, a chimeric antibody is one in which different portions of the antibody are derived from different animal species. For example, an antibody having a variable region derived from a murine monoclonal antibody paired with a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. For example, Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J., incorporated herein by reference in its entirety. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

  [110] Humanized forms of chimeric antibodies are made by substituting, for example, the complementarity determining regions of mouse antibodies within the human framework domain. See, for example, PCT Publication No. WO 92/22653. Humanized chimeric antibodies are preferably constant or variable regions other than complementarity determining regions (CDRs), and substantially or exclusively from non-human mammals, substantially or exclusively derived from the corresponding human antibody regions. Has a derived CDR.

[111] Artificial antibodies include scFv fragments, diabodies, triabodies, tetrabodies and mru, each having antigen-binding ability (Winter, G. and Milstein, C, 1991, Nature 349: 293). -299; see review by Hudson, PJ, 1999, Current Opinion in Immunology 11: 548-557). In a single chain Fv fragment (scFv), the V _H and V _L domains of the antibody are linked by a flexible peptide. Typically, this linker peptide is about 15 amino acid residues in length. If the linker is much smaller, for example 5 amino acids, a diabodies that are divalent scFv dimers are formed. When the linker is reduced to less than 3 amino acid residues, trimer and tetramer structures called triabodies and tetrabodies are formed. The minimal binding unit of an antibody is a CDR with sufficient specific recognition and binding, typically heavy chain CDR2, to be used separately. Such fragments are called molecular recognition units or mru. Some such mru may be linked together with a short linker peptide, thus forming an artificial binding protein with higher avidity than a single mru.

  [112] Functional equivalents of the present application also include modified antibodies, eg, antibodies that are modified by covalent attachment of any type of molecule to the antibody. For example, modified antibodies include, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, coupling to cellular ligands or other proteins, etc. Antibodies that have been modified are included. Such covalent linkage does not prevent the antibody from generating an anti-idiotypic response. These modifications may be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, the modified antibody may contain one or more non-classical amino acids.

  [113] Functional equivalents may be generated by exchanging different CDRs on different chains within different frameworks. Thus, for example, different heavy chain substitutions that can produce IgG1-4, IgM, IgA1-2, IgD, IgE antibody types and isotypes can also result in different classes of antibodies for a given set of CDRs. . Similarly, artificial antibodies within the scope of the present invention may be produced by embedding a given set of CDRs within a fully synthetic framework.

  [114] Functional equivalents by mutations, deletions and / or insertions within variable and / or constant region sequences adjacent to a particular set of CDRs using a wide variety of methods known in the art Can be easily produced.

  [115] Antibody fragments and functional equivalents of the invention include molecules that bind to CA6 to a detectable degree when compared to DS6 antibodies. For a detectable degree of binding, at least 10-100%, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90% of the binding capacity of the murine DS6 antibody to CA6. All values in the range of%, 95% or 99% are included.

Improved antibody [116] CDRs are most important for epitope recognition and antibody binding. However, changes may be made in residues comprising CDRs without interfering with the ability of the antibody to recognize and bind to its cognate epitope. For example, changes that do not affect epitope recognition and that further increase the binding affinity of the antibody for the epitope may be made.

  [117] Accordingly, also included within the scope of the present invention is an improvement in both murine and humanized antibodies, preferably with increased affinity, that also specifically recognizes and binds to CA6. It is a type.

[118] Several studies have examined the effects of introducing one or more amino acid changes at various positions in an antibody sequence on properties such as binding and expression levels based on knowledge of the primary antibody sequence. (Yang, WP et al., 1995, J. Mol.
Biol. , 254, 392-403; Et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, T .; J. et al. Et al., 1998, Nature Biotechnology, 16, 535-539).

[119] In these studies, oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or E. coli mutagenized strains Etc. have been used to generate equivalents of primary antibodies by altering the heavy and light chain gene sequences in the CDR1, CDR2, CDR3, or framework regions (Vaghhan, T. J.). 1998, Nature Biotechnology, 16, 535-539; Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, “Page Display of Peptides and Proteins”, Kay, B. Supervised by, Ac ademic Press). These methods of changing the primary antibody sequence have resulted in improved affinity of the secondary antibody (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 3576-3580; Boder USA, 97, 10701-10705; Davies, J. and Riechmann, L., 1996, Immunotechnology, 2, 169-179; , 1996, J. Mol. Biol., 256, 77-88; Short, M. K. et al., 2002, J. Biol.Chem., 277, 16365-16370; Furukawa, K. et al., 2001.
Biol. Chem. , 276, 27622-27628).

  [120] An anti-CA6 antibody with improved function, including improved affinity for CA6, using the antibody sequences described in the present invention by a similar command strategy that alters one or more amino acid residues of the antibody May be developed.

  [121] Improved antibodies also include antibodies with improved properties prepared by standard techniques of animal immunity, hybridoma formation, and selection for antibodies with specific properties.

Cytotoxic Agent [122] The cytotoxic agent used in the cytotoxic conjugates of the present invention is any that causes cell death, induces cell death, or in some way reduces cell viability. It may be a compound. Preferred cytotoxic agents include, for example, maytansinoids and maytansinoid analogs, taxoids, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs as defined below. These cytotoxic agents are conjugated to antibodies, antibody fragments, functional equivalents, improved antibodies and analogs thereof as disclosed herein.

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

Among the cytotoxic agents that can be used in the present invention to form maytansinoid [124] cytotoxic conjugates are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

  [125] Examples of suitable maytansinol analogs include those having modified aromatic rings and those having modifications at other positions. Such suitable maytansinoids are US Pat. 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; , 322, 348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545. .

[126] Specific examples of suitable analogs of maytansinol having a modified aromatic ring include:
[127] (1) C-19-dechloro (US Pat. No. 4,256,746) (prepared by LAH reduction of ansamitocin P2);
[128] (2) C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (US Pat. Nos. 4,361,650 and 4,307,016) (genus Streptomyces) (Prepared by demethylation using Streptomyces or Actinomyces, or dechlorination using LAH); and [129] (3) C-20-demethoxy, C-20-acyloxy (-OCOR ), +/- dechloro (US Pat. No. 4,294,757) (prepared by acylation with acyl chloride)
Is included.

[130] Specific examples of suitable analogues of maytansinol with other position modifications include:
[131] (1) C-9-SH (US Pat. No. 4,424,219) (prepared by reaction of H ₂ S or P ₂ S ₅ with maytansinol);
[132] (2) C-14-alkoxymethyl (demethoxy / CH ₂ OR) (US Pat. No. 4,331,598);
[133] (3) C-14-hydroxymethyl or acyloxymethyl (CH ₂ OH or CH ₂ OAc) (US Pat. No. 4,450,254) (prepared from Nocardia);
[134] (4) C-15-hydroxy / acyloxy (US Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces);
[135] (5) C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora);
[136] (6) C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); 137] (7) 4,5-deoxy (US Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol)
Is included.

In [138] preferred embodiment, the cytotoxic conjugates of the present invention, as the cytotoxic agent, previously, N ^{2 '-} deacetyl -N ^2' - (3- mercapto-1-oxopropyl) - called maytansine Furthermore, a thiol-containing maytansinoid (DM1) is used. DM1 has the following structural formula (I):

Indicated by.
[139] In another preferred embodiment, the cytotoxic conjugate of the present invention has, as a cytotoxic agent, a thiol-containing maytansinoid, N ^{2 ′} -deacetyl-N ^{2 ′} -(4-methyl-4-mercapto- 1-oxopentyl) -maytansine is utilized. DM4 has the following structural formula (II):

Indicated by.
[140] In further embodiments of the invention, other maytansines may be used, including thiol and disulfide containing maytansinoids bearing mono- or dialkyl substitutions on the carbon atom bearing the sulfur atom. These have an acylated amino acid side chain containing an acyl group bearing a sterically hindered sulfhydryl group at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl. Maytansinoids, wherein the carbon atom of the acyl group carrying the thiol functionality has one or two substituents, said substituents comprising CH3, C2H5, 1-10 carbon atoms. A linear or branched alkyl or alkenyl having a cyclic alkyl or alkenyl having 3 to 10 carbon atoms, a phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and further one of the substituents May be H and the acyl group is at least 3 between the carbonyl functionality and the sulfur atom. Having a linear length of the carbon atoms.

  [141] Such additional maytansines include compounds represented by formula (III):

In the formula:
Y ′ is (CR ₇ CR ₈ ) ₁ (CR ₉ = CR ₁₀ ) _p C = C _{q Ar} (CR ₅ CR ₆ ) _{m Du} (CR ₁₁ = CR ₁₂ ) _r (C = C) _s B _t ( CR ₃ CR ₄ ) _n CR ₁ R ₂ SZ
Indicate
In the formula:
R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, A phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radical, and furthermore R ₂ may be H;
A, B, D are cycloalkyl or cycloalkenyl, simple or substituted aryl or heteroaromatic or heterocycloalkyl radicals having 3 to 10 carbon atoms;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ , and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. Straight chain alkyl or alkenyl having 3 to 10 carbon atoms, branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radicals;
l, m, n, o, p, q, r, s, and t are each independently 0 or an integer of 1 to 5, provided that at any point in time, l, m, n, o, p , Q, r, s, and t are not zero; and Z is H, SR or —COR, wherein R is a linear alkyl or alkenyl having 1 to 10 carbon atoms, These include branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radicals.

[142] Preferred embodiments of formula (III) include:
R ₁ is H, R ₂ is methyl, and Z is H R ₁ and R ₂ are methyl and Z is H R ₁ is H, R ₂ is methyl, and Z is R ₁ and _{R 2} are methyl is -SCH _3, and Z is are compounds of the formula (III) is -SCH _3.

  [143] Such additional maytansines may also have the formula (IV-L), (IV-D), or (IV-D, L):

In the formula:
Y represents (CR ₇ CR ₈ ) _l (CR ₅ CR ₆ ) _m (CR ₃ CR ₄ ) _n CR ₁ R ₂ SZ
In the formula:
R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, A phenyl, substituted phenyl, or heteroaromatic or heterocycloalkyl radical, and furthermore R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 to 10 A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical having 5 carbon atoms;
l, m and n are each independently an integer from 1 to 5 and, in addition, n may be zero;
Z is H, SR or —COR, wherein R is a linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, a cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or Substituted aryl or heteroaromatic or heterocycloalkyl radicals; and May may represent a maytansinoid carrying a side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl Also included are the compounds shown.

[144] Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D, L) include:
R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ , and R ₈ are each H, l and m are each 1, n is zero, and Z is R ₁ and R ₂ that are H are methyl, R ₅ , R ₆ , R ₇ , R ₈ are each H, l and m are 1, n is zero, and Z is H R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ , and R ₈ are each H, l and m are each 1, n is zero, and Z is -SCH R ₁ and _{R 2} is ₃ is _{methyl, R 5, R 6, R 7} , R 8 are each H, l and m are 1, n is zero, and Z is - SCH ₃ in which formula (IV-L), include compounds of (IV-D) and (IV-D, L).

[145] Preferably, the cytotoxic agent is represented by formula (IV-L).
[146] These additional maytansines also have the formula (V):

In the formula:
Y represents (CR ₇ CR ₈ ) _l (CR ₅ CR ₆ ) _m (CR ₃ CR ₄ ) _n CR ₁ R ₂ SZ
In the formula:
R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, A phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radical, and furthermore R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 to 10 A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical having 5 carbon atoms;
l, m and n are each independently an integer from 1 to 5 and, in addition, n may be zero; and Z is H, SR or —COR, wherein R is Compounds represented by straight-chain alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical Is also included.

[147] Preferred embodiments of formula (V) include:
R1 is H, R2 is methyl, R5, R6, R7, and R8 are each H; l and m are each 1; n is zero; and Z is H R ₁ and R ₂ is methyl; R ₅ , R ₆ , R ₇ , R ₈ are each H, l and m are 1, n is zero; and Z is H R ₁ is H , R ₂ is methyl, R ₅ , R ₆ , R ₇ , and R ₈ are each H, l and m are each 1; n is zero; and Z is —SCH ₃ R _A formula wherein ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ , R ₈ are each H, l and m are 1, n is zero, and Z is —SCH ₃ The compound of V) is included.

  [148] Such additional maytansines may further include formula (VI-L), (VI-D), or (VI-D, L):

In the formula:
Y ₂ represents (CR ₇ CR ₈ ) _l (CR ₅ CR ₆ ) _m (CR ₃ CR ₄ ) _n CR ₁ R ₂ SZ ₂
In the formula:
R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, A phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radical, and furthermore R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear, cyclic alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radical having from 10 to 10 carbon atoms;
l, m and n are each independently an integer from 1 to 5 and, in addition, n may be zero;
Z ₂ is SR or COR, wherein R is a straight chain alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 1 to 10 carbon atoms, or a simple or substituted aryl Or a heteroaromatic or heterocycloalkyl radical; and May may include compounds represented by maytansinoids.

  [149] Such additional maytansine may also be a compound represented by formula (VII):

In the formula:
Y ₂ ′ is (CR ₇ CR ₈ ) _l (CR ₉ = CR ₁₀ ) p (C = C) _{q Ar} (CR ₅ CR ₆ ) _{m Du} (CR ₁₁ = CR ₁₂ ) _r (C = C) _s B _t (CR ₃ CR ₄ ) _n CR ₁ R ₂ SZ ₂
Indicate
In the formula:
R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, cyclic alkyl or alkenyl having 3 to 10 carbon atoms, A phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radical, and furthermore R ₂ may be H;
A, B, and D are each independently a cycloalkyl or cycloalkenyl, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical having 3 to 10 carbon atoms;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ , and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. Straight chain alkyl or alkenyl having 3 to 10 carbon atoms, branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radicals;
l, m, n, o, p, q, r, s, and t are each independently 0 or an integer of 1 to 5, provided that at any point in time, l, m, n, o, p , Q, r, s, and t are not zero; and Z ₂ is SR or —COR, wherein R is a linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 Also included are branched or cyclic alkyl or alkenyl having from -10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radicals.

[150] Preferred embodiments of formula (VII) include:
Included are compounds of formula (VII) wherein R1 is H and R2 is methyl.

[151] The maytansinoids described above may be conjugated to the anti-CA6 antibody DS6, or a homologue or fragment thereof, wherein the antibody is a C-3, C-14 hydroxymethyl, C The thiol or disulfide functionality present on the acyl group of the acylated amino acid side chain found in -15 hydroxy or C-20 desmethyl is linked to the maytansinoid and the acyl group of the acylated amino acid side chain is Straight chain alkyl or alkenyl having thiol or disulfide functionality located on a carbon atom having one or two substituents, wherein the substituent is CH ₃ , C ₂ H ₅ , 1-10 carbon atoms Branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl Is a heteroaromatic or heterocycloalkyl radical, and in addition, one of the substituents may be H, and the acyl group has a carbonyl functionality and a length of at least 3 carbon atoms between the sulfur atoms. It has a straight chain.

  [152] Preferred conjugates of the invention have formula (VIII):

In the formula:
Y ₁ ′ is (CR ₇ CR ₈ ) _l (CR ₉ = CR ₁₀ ) p (C = C) _{q Ar} (CR ₅ CR ₆ ) _{m Du} (CR ₁₁ = CR ₁₂ ) _r (C = C) _s B _t (CR ₃ CR ₄ ) _n CR ₁ R ₂ S-
Indicate
In the formula:
A, B, and D are each independently a cycloalkyl or cycloalkenyl, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical having 3 to 10 carbon atoms;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ , and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. Straight chain alkyl or alkenyl having 3 to 10 carbon atoms, branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical; and l, m, n, o, p, q, r, s, and t are each independently 0 or an integer from 1 to 5, provided that at any point in time, l, m, n, o, p, q, r, s, and At least two of t include the anti-anti-CA6 antibody DS6, or a homologue or fragment thereof, conjugated to a non-zero maytansinoid.

[153] Preferably, R ₁ is H and R ₂ is methyl, or R ₁ and R ₂ are methyl.
[154] Even more preferred conjugates of the invention are of formula (IX-L), (IX-D), or (IX-D, L):

In the formula:
Y ₁ represents (CR ₇ CR ₈ ) _l (CR ₅ CR ₆ ) _m (CR ₃ CR ₄ ) _n CR ₁ R ₂ S-
In the formula:
R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, A phenyl, substituted phenyl, heteroaromatic or heterocycloalkyl radical, and furthermore R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 to 10 A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl or heteroaromatic or heterocycloalkyl radical having 5 carbon atoms;
l, m and n are each independently an integer from 1 to 5 and, in addition, n may be zero; and May may be C-3, C-14 hydroxymethyl, C-15 hydroxy or It comprises an anti-CA6 antibody DS6, or a homologue or fragment thereof, conjugated to a maytansinoid that exhibits maytansinol carrying a side chain at C-20 desmethyl.

[155] Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D, L) include:
R ₁ is H and R ₂ is methyl, or R ₁ and R ₂ are methyl,
R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ , and R ₈ are each H; l and m are each 1; n is 0;
R ₁ and R ₂ are methyl; R ₅ , R ₆ , R ₇ , and R ₈ are each H; l and m are 1; and n is 0 (IX-L), (IX -D) and (IX-D, L) compounds are included.

[156] Preferably, the cytotoxic agent is represented by formula (IX-L).
[157] Further preferred conjugates of the invention are represented by formula (X):

Wherein the substituent comprises an anti-CA6 antibody DS6, or a homologue or fragment thereof, conjugated to a maytansinoid as defined for formula (IX) above.

[158] Particularly preferred is that R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1 and n Is any of the above compounds wherein 0 is 0.

[159] Even more particularly preferred are R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1 and n is 0 Any of the above compounds.

[160] Furthermore, L-aminoacyl stereoisomers are preferred.
[161] Each maytansinoid described in pending US patent application Ser. No. 10 / 849,136, filed May 20, 2004 can also be used in the cytotoxic conjugates of the present invention. The entire disclosure of US patent application Ser. No. 10 / 849,136 is incorporated herein by reference.

Disulfide-containing linking group [162] In order to link a maytansinoid to a cell binding agent such as a DS6 antibody, the maytansinoid includes a linking group. The linking moiety contains chemical bonds at specific sites that allow the release of fully active maytansinoids. Suitable chemical bonds are well known in the art and include disulfide bonds, acid labile bonds, photodissociative bonds, peptidase labile bonds and esterase labile bonds. Preferred is a disulfide bond.

[163] The linking moiety also includes a reactive chemical group. In preferred embodiments, the reactive chemical group may be covalently linked to the maytansinoid via a disulfide bond linking moiety.
[164] Particularly preferred reactive chemical groups are N-succinimidyl esters and N-sulfosuccinimidyl esters.

  [165] Particularly preferred maytansinoids comprising a linking moiety containing a reactive chemical group, wherein the linking moiety contains a disulfide bond and the chemically reactive group comprises N-succinimidyl or N-sulfosuccinimidyl ester. C-3 ester of maytansinol and its analogs.

  [166] Many positions on the maytansinoid can serve as positions that chemically link the linking moieties. For example, the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy, and the C-20 position with a hydroxy group are all expected to be useful. The However, the C-3 position is preferred, and the C-3 position of maytansinol is particularly preferred.

  [167] While the synthesis of maytansinol esters having linking moieties will be described in terms of disulfide bond-containing linking moieties, those skilled in the art will also understand linking moieties with other chemical bonds (such as those described above) It will be understood that other maytansinoids can be used in the present invention. Specific examples of other chemical bonds include acid labile bonds, photodissociative bonds, peptidase labile bonds and esterase labile bonds. The disclosure of US Pat. No. 5,208,020, incorporated herein, describes the production of maytansinoids possessing such binding.

  [168] Synthesis of maytansinoids and maytansinoid derivatives having a disulfide moiety bearing a reactive group is incorporated herein by reference, US Pat. Nos. 6,441,163 and 6,333, respectively. 410, as well as US application Ser. No. 10 / 161,651.

  [169] A reactive group-containing maytansinoid such as DM1 is reacted with an antibody such as a DS6 antibody to produce a cytotoxic conjugate. These conjugates may be purified by HPLC or by gel filtration.

  [170] Several excellent schemes for producing such antibody maytansinoid conjugates are described in US Pat. No. 6,333,410, as well as US applications, each incorporated herein in its entirety. 09 / 867,598, 10 / 161,651 and 10 / 024,290.

  [171] Generally, an antibody solution in an aqueous buffer may be incubated with a molar excess of maytansinoid having a disulfide moiety carrying a reactive group. The reaction mixture may be quenched by the addition of excess amine (ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate may then be purified by gel filtration.

  [172] By measuring the ratio of absorbance at 252 nm and 280 nm spectrophotometrically, the number of maytansinoid molecules bound per antibody molecule can be determined. An average of 1-10 maytansinoid molecules / antibody molecules is preferred.

[173] Maytansinoid drug-antibody conjugates can be evaluated in vitro for their ability to inhibit the growth of a variety of undesirable cell lines. For example, cell lines such as the human epidermoid carcinoma cell line A-431, the human small cell lung cancer cell line SW2, the human breast tumor cell line SKBR3 and the Burkitt lymphoma cell line Namalwa can be used to assess the cytotoxicity of these compounds. Easy to use. Cells to be evaluated can be exposed to the compound for 24 hours and cell viability can be measured in a direct assay by known methods. IC ₅₀ values can then be calculated from the assay results.

PEG-containing linking groups [174] maytansinoids may also be linked to cell binding agents using PEG linking groups, as shown in US Application No. 10 / 024,290. These PEG linking groups are soluble both in water and in non-aqueous solvents, and may be used to link one or more cytotoxic agents to the cell binding agent. A typical PEG linking group includes a heterobifunctional that attaches to a cytotoxic and cell binding agent at the opposite end of the linker through a functional sulfhydryl or disulfide group at one end and an active ester at the other end. Sex PEG linkers are included.

  [175] As a general example of the synthesis of cytotoxic conjugates using PEG linking groups, reference is again made to US Application No. 10 / 024,290 for specific details. The synthesis begins with the reaction of one or more cytotoxic agents possessing a reactive PEG moiety and a cell binding agent, which reaction is a terminally active ester of each reactive PEG moiety by an amino acid residue of the cell binding agent. Resulting in a cytotoxic conjugate comprising one or more cytotoxic agent covalently attached to the cell binding agent through a PEG linking group.

Taxane [176] The cytotoxic agent used in the cytotoxic conjugates of the present invention may also be a taxane or a derivative thereof.

  [177] Taxanes are a family of compounds that include the cytotoxic natural product paclitaxel (taxol) and the semi-synthetic derivative docetaxel (taxotere), these two compounds being widely used in the treatment of cancer. Yes. Taxanes are spindle toxins that inhibit tubulin depolarization and cause cell death. Docetaxel and paclitaxel are useful agents for cancer treatment, but their antitumor activity is limited because they are nonspecifically toxic to normal cells. Furthermore, compounds such as paclitaxel and docetaxel are themselves not powerful enough for use in conjugates of cell binding agents.

  [178] Preferred taxanes for use in preparing cytotoxic conjugates are those of formula (XI):

It is.
[179] A method for synthesizing taxanes that can be used in the cytotoxic conjugates of the present invention, as well as methods for conjugating taxanes to cell binding agents such as antibodies, are described in US Pat. No. 5,416, No. 064, No. 5,475,092, No. 6,340,701, No. 6,372,738 and No. 6,436,931, and U.S. Application Nos. 10 / 024,290 and 10/144. No. 042, No. 10 / 207,814, No. 10 / 210,112 and No. 10 / 369,563.

CC-1065 analog [180] The cytotoxic agent used in the cytotoxic conjugates according to the invention may also be CC-1065 or a derivative thereof.

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

  [182] The cytotoxicity of CC-1065 has been correlated with its alkylating activity and its DNA binding or DNA insertion activity. These two activities belong to separate parts of the molecule. Thus, the alkylating activity is contained in the cyclopropyropyrrole indole (CPI) subunit and the DNA binding activity belongs in the two pyrroloindole subunits.

  [183] CC-1065 has certain attractive features as a cytotoxic agent, but has limitations in therapeutic use. Administration of CC-1065 to mice causes delayed hepatotoxicity leading to death at day 50 after a single intravenous dose of 12.5 μg / kg {V. L. Reynolds et al., J. MoI. Antibiotics, XXIX, 319-334 (1986)}. This has spurred efforts to develop analogs that do not cause delayed toxicity and the synthesis of simpler analogs modeled on CC-1065 has been described {M. A. Warpehoski et al., J. MoI. Med. Chem. , 31, 590-603 (1988)}.

  [184] In another series of analogs, the CPI moiety was replaced by a cyclopropabenzindole (CBI) moiety {D. L. Boger et al. Org. Chem. , 55, 5823-5833, (1990), D.C. L. Boger et al., BioOrg. Med. Chem. Lett. , 1, 115-120 (1991)}. These compounds maintain the high in vitro strength of the parent drug in mice without causing delayed toxicity. Like CC-1065, these compounds are alkylating agents that bind to the minor groove of DNA in a covalent manner, resulting in cell death. However, clinical evaluation of the most promising analogs, adzelesin and calzeresin, led to disappointing results {B. F. Foster et al., Investigative New Drugs, 13, 321-326 (1996); Wolff et al., Clin. Cancer Res. , 2, 1717-1723 (1996)}. These drugs exhibit poor therapeutic effects due to their high systemic toxicity.

  [185] Through targeted delivery to the tumor site, the therapeutic efficacy of CC-1065 analogs is greatly enhanced by altering the in vivo distribution, resulting in lower toxicity to untargeted tissue and thus lower systemic toxicity It is also possible to improve. To achieve this goal, conjugates of cell binding agents and analogs and derivatives of CC-1065 have been described that specifically target tumor cells {US Pat. No. 5,475,092; 5,585,499; 5,846,545}. These conjugates typically show high target-specific cytotoxicity in vitro and show exceptional antitumor activity in a human tumor xenograft model in mice {R. V. J. et al. Chari et al., Cancer Res. , 55, 4079-4084 (1995)}.

  [186] Methods for synthesizing CC-1065 analogs that can be used in the cytotoxic conjugates of the present invention, as well as methods for conjugating analogs to cell binding agents such as antibodies, are described in US Patents. No. 5,475,092, No. 5,846,545, No. 5,585,499, No. 6,534,660 and No. 6,586,618, and U.S. Application No. 10 / 116,053. No. and 10 / 265,452 are described in detail.

Other drugs [187] methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulinsin and tubulincin analogs, duocarmycin and duocarmycin analogs, dolastatin And drugs such as dolastatin analogs are also suitable for preparing the conjugates of the invention. Alternatively, the drug molecule may be linked to the antibody molecule through a mediator carrier molecule such as serum albumin. Doxorubicin and daunorubicin compounds can also be useful cytotoxic agents, as described in US 09/740991.

Therapeutic Composition [188] The present invention also provides:
Also provided are therapeutic compositions comprising (a) an effective amount of one or more cytotoxic conjugates, and (b) a pharmaceutically acceptable carrier.

  [189] Similarly, the present invention provides a method for inhibiting the growth of a selected cell population, wherein a target cell, or a tissue containing the target cell is treated with an effective amount of a cytotoxic conjugate, or cytotoxicity. There is provided a method comprising contacting a therapeutic agent comprising a sex conjugate, alone or in combination with other cytotoxic or therapeutic agents.

[190] The present invention also includes a method for treating a subject having cancer using the therapeutic composition of the present invention.
[191] Cytotoxic conjugates can be evaluated for in vitro strength and specificity by the methods described previously (eg, RVJ Chari et al., Cancer Res. 55: 4079-4084). (See 1995). Again, antitumor activity can be assessed in human tumor xenograft models in mice by the methods described previously (see, eg, Liu et al., Proc. Natl. Acad. Sci. 93: 8618-8623 (1996)). See).

  [192] Suitable pharmaceutically acceptable carriers are well known and can be determined by one of ordinary skill in the art as warranted by the clinical situation. As used herein, a carrier includes diluents and excipients.

  [193] Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco's phosphate buffered saline, with or without about 1 mg / ml to 25 mg / ml human serum albumin. Water, pH˜7.4, (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)), and (3) 5% (w / v) dextrose; Such carriers may also contain an antioxidant such as tryptamine and a stabilizer such as Tween 20.

[194] A method for inhibiting the growth of a selected cell population is described in vitro, in
It can be performed in vivo or ex vivo. As used herein, inhibition of proliferation means slowing of cell growth, reduced cell viability, causing cell death, cell lysis, and induction of cell death, whether short or long term. .

  [195] Examples of in vitro use include treatment of autologous bone marrow prior to transplantation into the same patient to kill diseased or malignant cells; kill eligible T cells and graft versus host disease (GVHD) Treatment of bone marrow prior to transplantation to prevent; kill all cells except the desired variant that does not express the target antigen; or treatment of the cell culture to kill the variant that expresses the unwanted antigen included.

[196] Conditions for nonclinical in vitro use are readily determined by those of ordinary skill in the art.
[197] Examples of clinical ex vivo use include removing tumor cells or lymphocytes from bone marrow prior to autotransplantation or preventing graft-versus-host disease (GVHD) in cancer treatment or in autoimmune disease treatment To remove T cells or other lymphocytes from autologous or allogeneic bone marrow or tissue prior to transplantation. Treatment may be performed as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum supplemented with the cytotoxic agents of the present invention. Concentrations range from about 10 μM to 1 pM and are incubated at about 37 ° C. for about 30 minutes to about 48 hours. The exact conditions of concentration and incubation time, ie dose, are readily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with medium containing serum and i. v. Return to patient by injection. In situations where patients receive other treatments such as excision chemotherapy or whole body irradiation during bone marrow collection and reinfusion of treated cells, the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical devices May be.

  [198] For clinical in vivo use, the cytotoxic conjugates of the invention are supplied as a solution and tested for sterility and endotoxin levels. An example of a suitable protocol for cytotoxic conjugate administration is as follows. Conjugates weekly for 4 weeks, i. v. Administer as a bolus. The bolus dose is administered in 50-100 ml of physiological saline, to which 5-10 ml of human serum albumin may be added. The dosage is i. v. 10 μg to 100 mg per dose (range of 100 ng to 1 mg / kg per day). More preferably, the dosage will be in the range of 50 μg to 30 mg. Most preferably, the dosage will range from 1 mg to 20 mg. After 4 weeks of treatment, the patient may continue to receive treatment weekly. The particular clinical protocol may be determined by one of ordinary skill in the art with regard to route of administration, excipients, diluents, dosage, time, etc., as the clinical situation warrants.

  [199] Examples of medical conditions treatable according to in vivo or ex vivo methods that kill selected cell populations include those in which CA6 is expressed, eg, lung, breast, colon, prostate, kidney, pancreas, ovary Include any type of malignant tumor, including cancer of the cervix and lymphoid organs, osteosarcoma, synovial cancer, sarcoma or carcinoma, and other cancers in which the CA6 glycotope is predominantly expressed Autoimmune diseases such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; transplant rejection such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; Graft-versus-host disease; viral infections such as mV infection, HIV infection, AIDS; and parasitic infections such as rumbled trichinosis, amebiasis, schistosomiasis, and general It includes others as determined by art.

Kit [200] The present invention also includes kits comprising, for example, the cytotoxic conjugates described, and instructions for use of the cytotoxic conjugates to kill a particular cell type. The instructions for use may include instructions for using the cytotoxic conjugate in vitro, in vivo or ex vivo.

  [201] Typically, the kit will have compartments containing cytotoxic conjugates. The cytotoxic conjugate may be lyophilized, liquid, or any other type acceptable to be included in the kit. The kit also includes additional elements necessary to perform the methods described in the instructions in the kit, such as a sterile solution for reconstitution of the lyophilized powder, a cytotoxic conjugate prior to administration to the patient. Additional agents for combination with, and tools useful for administering the conjugate to the patient may also be included.

Further Embodiments [202] The present invention further provides monoclonal antibodies, humanized antibodies and epitope binding fragments thereof that are further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radiolabel, phosphor, chromophore, contrast agent or metal ion.

  [203] A diagnostic method of administering the labeled humanized antibody or epitope-binding fragment thereof to a subject suspected of having cancer and measuring or monitoring the label distribution within the subject also comprises provide.

Examples [204] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments.

Example 1 Identification of Antigen Positive and Negative Cell Lines by Flow Cytometry Binding Assay [205] Flow cytometric analysis was used to localize the DS6 epitope, CA6, to the cell surface. With the exception of OVCAR5 (Kearse et al., Int. J. Cancer 88 (6): 866-872 (2000)), OVCAR8 and IGROV1 cells (M. Seiden, Massachusetts General Hospital), human cell lines were treated with American type culture. Obtained from the collection (ATCC). Hereinafter referred to as media, 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin (Cambrex Bio Science, Rockland, Maine) and 10% v / v fetal calf serum (Atlas Biologicals, Colorado) All cells were grown in RPMI 1640 supplemented with Fort Collins. Cells were maintained in a 37 ° C., 5% CO ₂ humidified incubator.

[206] 96-well plates, FACS buffer (2% goat serum, RPMI) and DS6 antibody continuous dilution prepared in 3-4 hours incubation of cells (1～2X10 ^-5 cells / well) on ice did. Cells were spun down in a tabletop centrifuge for 5 minutes at 1500 rpm and 4 ° C. After removing the medium, the wells were refilled with 150 μl FACS buffer. The washing process was then repeated. FITC-labeled goat anti-mouse IgG (Jackson Immunoresearch) was diluted 1: 100 in FACS buffer and incubated with cells for 1 hour on ice. The plate was covered with foil to prevent photobleaching of the signal. After two washes, the cells were fixed with 1% formaldehyde and analyzed on a flow cytometer.

[207] As predicted from tumor immunohistochemistry, the CA6 epitope is found primarily in cell lines of ovarian, breast, cervical, and pancreatic origin (Table 3). However, several cell lines from other tumor types showed limited CA6 expression. DS6 antibody binds with a _{K D} apparent 135.6PM (in PC-3 cells, Table 3). The maximum mean fluorescence (Table 3) of the binding curve (FIG. 1) in the antigen positive cell line suggests relative antigen density.
Table 3

^* Average maximum relative average fluorescence

Example 2: Characterization of the DS6 epitope [208] Dot blot of CA6 positive cell lysate (Caov-3) digested with proteolytic (pronase and proteinase K) and / or glycolytic (neuraminidase and periodic acid) treatments Were analyzed for the properties of the DS6 antigen, CA6. For positive controls, other antibodies recognizing various epitope species were tested against lysates of antigen positive cell lines (Caov-3 and CM1; Colo205 and C242; SKMEL28 and R24). CM1 is an antibody that recognizes the protein epitope of the variable number tandem repeat domain (VNTR) of Muc-1, and thus provides a control for the protein epitope. C242 binds to a novel colorectal cancer-specific sialic acid-dependent glycotope (CanAg) on Muc-1, which provides a control for glycotopes on proteins. R24 binds to the GD3 ganglioside specific for melanoma and thus provides a control for glycotopes on non-protein scaffolds.

[209] Caov-3, Colo205, and SKMEL28 cells were plated in 15 cm tissue culture plates. The medium (30 ml / plate) was renewed the day before lysis. Modified RIPA buffer (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 Was pre-cooled on ice. After aspirating the medium from the plate, the cells were washed twice with 10 ml of cold PBS. All subsequent steps were performed on ice and / or in a cold room at 4 ° C. After aspirating the last wash of PBS, lysis buffer (RIPA buffer with fresh addition of protease inhibitors to final concentrations of 1 mM PMSF, 1 μM pepstatin A, 10 μg / ml leupeptin, and 2 μg / ml aprotinin) 1 Cells were lysed in 2 ml. Using a cell lifter, the lysate was scraped off the plate and triturated by pipetting the suspension up and down (5-10 times) with an 18G needle. The lysate was spun for 10 minutes and then centrifuged in a microcentrifuge for 10 minutes at maximum power (13K rpm). The pellet was discarded and the supernatant was then assayed using the Bradford Protein Assay Kit (Biorad).

[210] Lysate (2 μl) was pipetted directly onto a dry 0.2 μm nitrocellulose membrane. The spot was allowed to air dry for approximately 30 minutes. The membrane was divided into pieces each containing a single spot. Pronase (1 mg / ml enzyme, 50 mM Tris pH 7.5, 5 mM CaCl ₂ ), proteinase K (1 mg / ml enzyme, 50 mM Tris
spot in the presence of pH 7.5, 5 mM CaCl ₂ ), neuraminidase (20 mU / ml enzyme, 50 mM sodium acetate pH 5, 5 mM CaCl ₂ , 100 μg / ml BSA) or periodic acid (20 mM, 0.5 M sodium acetate pH 5) Was incubated at 37 ° C. for 1 hour. Reagents were purchased from Roche (enzyme) and VWR (periodic acid). Membranes were washed (5 minutes) in T-TBS wash buffer (0.1% Tween 20, 1 × TBS), blocked in blocking buffer (3% BSA, T-TBS) for 2 hours at room temperature and blocked Incubated overnight with 2 μg / ml primary antibody (ie DS6, CM1, C242, R24) in buffer. The membrane was washed 3 times for 5 minutes in T-TBS and then HRP conjugated goat anti-mouse (or human) IgG secondary antibody (Jackson Immunoresearch; 1: 2000 dilution in blocking buffer) and room temperature And incubated for 1 hour. The immunoblot was washed 3 times and developed using the ECL system (Amersham).

  [211] The immunoblot of the digested control lysate (Figure 2) shows that the proteolytic treatment destroys the CM1 signal while the glycolytic digestion signal is affected as expected for antibodies recognizing protein epitopes. Showed that he did not. The C242 signal was destroyed by either proteolytic or glycolytic processes, as expected for antibodies that recognize glycotopes found on proteins. The R24 signal that was not affected by the proteolytic treatment disappeared with neuraminidase treatment or periodate treatment, as expected for antibodies that recognize gangliosides. DS6 immunoblot of digested Caov-3 lysate showed loss of signal upon treatment with either proteolytic or glycolytic compounds. Thus, like C242, DS6 binds to a carbohydrate epitope on the proteinaceous core. Furthermore, the signal in the DS6 immunoblot was sensitive to neuraminidase treatment. Thus, like CanAg, CA6 is a sialic acid dependent glycotope.

  [212] To confirm that CA6 is carbohydrate, the Caov-3 lysate was spotted onto a PVDF membrane and the chemical deglycosylating agent, trifluoromethanesulfonic acid (TFMSA), under nitrogen at ambient temperature For 5 minutes. The blot was washed with T-TBS and immunoblotted with either CM1 or DS6 (FIG. 3). The DS6 signal was disrupted by acid treatment, providing further evidence that CA6 is a glycotope. The CM1 signal is enhanced upon TFSMA treatment, indicating that acid treatment does not affect the protein on the filter and suggests that glycolysis exposes the protein epitope recognized by CM1.

[213] To further elucidate the structure of the carbohydrate to which CA6 belongs,
-Digested with glycanase, O-glycanase, and / or sialidase (Figure 4). Caov-3 cell lysate (100 μg, 30 μl) was incubated for 5 minutes at 100 ° C. with 2.5 μl of denaturing buffer (Glyko) containing SDS and β-mercaptoethanol. The denatured lysate was then digested with 1 μl N-glycanase, O-glycanase, and / or sialidase A (Glyko) for 1 hour at 37 ° C. The digested lysate was then spotted on nitrocellulose (2 μl) and immunoblotted as described above.

  [214] N-glycanase had no apparent effect on the DS6 immunoblot signal. However, samples digested with sialidase did not produce any signal. Since the O-glycanase cannot digest the sialylated O-linked carbohydrate without pretreatment with sialidase, the DS6 signal of the sample processed with O-glycanase alone will not be affected. In contrast, N-glycanase does not require pretreatment with any glycoside enzyme because of its activity. The fact that N-glycanase treatment does not affect the DS6 signal suggests that the CA6 epitope is most likely present on the sialylated O-linked carbohydrate chain.

Example 3: Elucidation of antigens where CA6 epitopes are found [215] DS6 immunoprecipitates were analyzed by SDS-PAGE and Western blotting to identify antigens where CA6 sialoglycotopes were found. Cell lysate supernatant (1 ml / sample; 3-5 mg protein) was pre-cleaned with protein G beads (30 μl) and equilibrated with 1 ml RIPA buffer at 4 ° C. for 1-2 hours with rotation. All subsequent steps were performed on ice and / or in a cold room at 4 ° C. The pre-cleaned beads were dropped by simply rotating (2-3 seconds) in a microcentrifuge. The pre-cleaned supernatant was transferred to a new tube and incubated with 2 μg DS6 overnight with rotation. Fresh, equilibrated protein G beads (30 μl) were added to the lysate and incubated for 1 hour with rotation. The bead-lysate suspension was dropped by simple rotation in a microcentrifuge and optionally a sample of the lysate after immunoprecipitation was taken. The beads were washed 5-10 times with 1 ml RIPA buffer.

[216] The immunoprecipitated DS6 sample was then added to 30 μl neuraminidase (20 mU neuraminidase (Roche), 50 mM sodium acetate pH 5, 5 mM CaCl ₂ , 100 μg / ml BSA) or 30 μl periodic acid (20 mM periodic acid (VWR)). And 0.5 M sodium acetate pH 5) and digested at 37 ° C. for 1 hour. They were then resuspended in 30 μl of 2 × sample loading buffer (containing β-mercaptoethanol). The beads were boiled for 5 minutes and the loading buffer supernatant was loaded onto a 4-12% or 4-20% Tris-glycine gel (Invitrogen). Gels were run in Laemmli electrophoresis buffer at 125V for 1.5 hours. Gel samples were transferred overnight at 20 mA onto a 0.2 μm nitrocellulose membrane (Invitrogen) using a Mini Trans-blot transfer device (Biorad). Membranes were immunoblotted with DS6 as described above in Example 2.

[217] Alternatively, immunoprecipitated beads were first denatured and then enzymatically digested with N-glycanase, O-glycanase and / or sialidase A (Glyko). The beads were resuspended in 27 μl incubation buffer and 2 μl denaturing solution (Glyko) and incubated at 100 ° C. for 5 minutes. After cooling to room temperature, detergent solution (2 μl) was added and samples were incubated with 1 μl N-glycanase, O-glycanase, and / or sialidase A for 4 hours at 37 ° C. After adding 5 × sample loading buffer (7 μl), the sample was boiled for 5 minutes. Sample is SDS-
PAGE and immunoblotting as described above.

  [218] DS6 immunoprecipitates a> 250 kDa protein band that can be found in antigen-positive cell lysates (FIGS. 5A, B and C). In some cell lines (ie T-47D) doublets are observed. The> 250 kDa band disappears in Caov-3 immunoprecipitates treated with neuraminidase or periodic acid (FIGS. 5A and B), suggesting that the CA6 epitope belongs on the> 250 kDa band. The> 250 kDa band was also shown to be insensitive to N-glycanase treatment of immunoprecipitates, consistent with CA6 belonging to O-linked carbohydrate (FIG. 5F). Further supporting that the 250 kDa band is the CA6 antigen is the fact that DS6 does not immunoprecipitate these bands from DS6 antigen negative cells (FIGS. 5D and E).

  [219] Several lines of evidence suggested that the CA6 antigen is Mucl. Due to its high molecular weight and sensitivity to O-linked carbohydrate-specific glycolytic enzymes, it appears likely that the CA6 antigen is a mucin. Mucin overexpression is well characterized in tumors, especially breast and ovarian tumors, which is consistent with the main tumor reactivity of DS6. Furthermore, CA6 is not as sensitive to perchloric acid precipitation as CanAg (sialoglycotope on Muc1), suggesting that the CA6 antigen is highly O-glycosylated. In several DS6-expressing cell lines, DS6 immunoprecipitated> 250 kDa doublets suggested that CA6 was Muc1. A characteristic of Muc1 in humans is the presence of two separate Muc1 alleles that differ in the number of tandem repeats, which results in the expression of two Muc1 proteins of different molecular weight.

  [220] To test whether CA6 is found on Muc1, DS6 immunoprecipitates from Caov-3 lysates were subjected to SDS-PAGE and immunoblotted with either DS6 or Muc1 VNTR antibody, CM1 did. As can be seen in FIG. 6A, CM1 reacts strongly with the> 250 kDa band that is immunoprecipitated by DS6. In FIG. 6B, DS6 and CM1 immunoprecipitates from HeLa cell lysates show the same> 250 kDa doublet when immunoblotted with either DS6 or CM1. These results indicate that the CA6 epitope is indeed located on the Muc-1 protein. The DS6 doublet found in HeLa (and T-47D) cells can be explained by the fact that Muc-1 expression is directed by distinct alleles with different numbers of tandem repeats.

[221] CM1 and DS6 bind to the same Muc-1 protein, but these are distinct epitopes. Chemical deglycosylation of Caov-3 lysate dot blots with trifluoromethanesulfonic acid (TFMSA) abolished the DS6 signal (FIG. 3). However, this same treatment enhanced the CM1 signal. Deglycosylation may have exposed a hidden epitope of the CM1 antibody. Furthermore, comparison of DS6 and CM1 flow cytometry binding results (Table 4) shows that the CA6 epitope is not present on all cells expressing Muc1. It is interesting to note that the CA6 epitope is not expressed on Colo205, a cell line known to express high levels of Mucl CanAg sialoglycotopes (Table 3).
Table 4

^* MMF = maximum average relative fluorescence

Example 4: Quantitative analysis of sheed CA6 epitopes [222] Since CA6 epitopes belong to Muc1, a molecule known to drop into the bloodstream in many cancer patients, these levels are DS6. A quantitative approach was taken to determine if antibody therapy was prohibitive. Binding of circulating antibodies to the antigen is thought to lead to rapid clearance of immune complexes from the blood. If a significant portion of the administered antibody dose is cleared rapidly from the circulation, the amount that reaches the tumor is likely to decrease, resulting in a decrease in the anti-tumor activity of the antibody therapeutic. When the antibody is conjugated to a very potent cytotoxic compound, rapid clearance of the conjugate can potentially increase nonspecific toxicity. Thus, in the case of antibody-small drug conjugates such as DS6-DM1, high levels of shed antigen can be expected to reduce anti-tumor effects and increase dose limiting toxicity.

[223] Recent clinical trials of antibody therapeutics have yielded information on the effect of dropped antigen concentration on pharmacokinetics. For example, in a clinical trial using trastuzumab (Herceptin), an antibody used to treat metastatic breast cancer that expresses her2 / neu, the pharmacokinetics of trastuzumab clearance is when the shedding Her2 / neu level is less than 500 ng / ml Have been shown to be unmodified (Pegram et al., J. Clin. Oncol.
16 (8): 2659-71 (1998)). Assuming the molecular weight of shed Her2 / neu is 110,000 daltons, a molar concentration of shed Her2 / neu of less than 4.5 nM appears to have little effect on pharmacokinetics.

[224] In another example, a clinical trial using cantuzumab mertansine (huC242-DMl) showed that there was no correlation between pretreatment dropout CanAg (C242 epitope) levels and antibody clearance pharmacokinetics. (Tolcher et al., J. Clin. Oncol. 21 (2): 211-22 (2003)). The CanAg epitope, like the CA6 epitope recognized by DS6, is a unique tumor-specific O-linked sialoglycotope on Muc1. However, the CanAg epitope is heterogeneous and difficult to quantify in moles. In the general population, Mucl alleles vary in length depending on the number of tandem repeats in the variable number tandem repeat (VNTR) domain. There are several sites for O-linked glycosylation in each tandem repeat. In addition to the complexity of CanAg expression, there is intercellular variation in innate glycosyltransferase activity. Thus, there can be a broad range of CanAg epitopes per Muc molecule, even in a single patient. Furthermore, the ratio of CanAg epitopes per Muc molecule will vary across patient populations. For this reason, the missing Muc1 containing the CanAg epitope is captured by C242,
Dropped CanAg in the serum sample is then measured by sandwich ELISA, detected by the biotinylated C242 / streptavidin HRP system. Dropped CanAg is quantified in standardized units proportional to the number of epitopes per ml of serum rather than by the molar concentration of Mucl. Similarly, a similar situation arises regarding quantification of the missing CA6 epitope. In contrast, for trastuzumab, there is only one epitope per shed her2 / neu molecule, and quantification of shed antigen is greatly simplified.

  [225] Developed a method to obtain molar concentrations of complex shed epitopes such as sialoglycotopes on Muc1 to correlate CA6 shed epitope levels with those seen in clinical trials with trastuzumab and cantuzumab martansine did. First, a simple sandwich ELISA assay for DS6 was established. A representative assay is shown in FIG. 7A. DS6 was used to capture Muc1 with the CA6 epitope. Since each Mucl molecule has multiple CA6 epitopes, biotinylated DS6 was also used as a tracer antibody. Using ABTS as a substrate, biotinylated DS6 bound to captured CA6 was detected by streptavidin-HRP. CA6 epitopes were captured from ovarian cancer patient sera or from standards derived from a commercially available Mucl test kit (CA15-3) used to monitor shed Muc1 in breast cancer patients. DS6 units / ml was arbitrarily set equal to CA15-3 standard units / ml.

  [226] In FIG. 7B, the results of a DS6 sandwich ELISA using the CA15-3 standard are shown. The resulting curve is very similar to that obtained with the CA15-3 standard in the CA15-3 assay. In order to convert DS6 units / ml to the molar concentration of CA6, a standard curve for biotinylated DS6 is required, which converts the signal to a picogram of DS6. Assuming a one-to-one stoichiometry between the CA6 epitope and biotinylated DS6 antibody and the molecular weight of biotinylated DS6 is 160,000 daltons, the number of moles of CA6 captured per added sample volume May be calculated.

  [227] In FIGS. 8A and B, two alternative means of generating a standard curve for biotinylated DS6 are shown. In FIG. 8A, a goat anti-mouse IgG polyclonal antibody is used to capture biotinylated DS6, which is then detected in the same manner as used in the sandwich ELISA assay shown in FIG. In the method shown in FIG. 8B, biotinylated DS6 is plated directly on an ELISA plate and detected as in FIG. 8A. As seen in FIG. 8C, the biotinylated DS6 standard curves generated by each method are in good agreement.

[228] In Table 5, analysis of ovarian cancer patient serum samples for various dropped antigens is shown. In general, treatment of ovarian cancer patients is monitored by measuring dropout CA125 units / ml using a CA125 ELISA. Serum samples are used to provide CA125 status. In general, the treatment of breast cancer patients is monitored using CA15-3 ELISA using capture and detection antibodies that recognize different epitopes than those recognized by DS6 and measuring units / ml of shed Muc1. . In Table 5, CA15-3 is measured in serum samples of ovarian cancer patients.
Table 5

Measured by ¹ commercial ELISA kit
² Measured according to commercial CA15-3 standard (1 CA15-3 U = 1 DS6 U)
³ goat anti-mouse IgG and biotin-DS6 standard curve
⁴ Biotin-DS6 standard curve

  [229] For the CA15-3 values reported in Table 5, a commercially available CA15-3 enzyme immunoassay kit from CanAg Diagnostics was used. For DS6 units / ml, a standard curve was generated in a DS6 sandwich ELISA using the CA15-3 standard (from the CA15-3 enzyme immunoassay kit from CanAg Diagnostics). DS6 units / ml was arbitrarily set equal to CA15-3 units / ml. In the last two columns, picomolar (pM) shedding CA6 was calculated using the biotinylated DS6 standard curve shown in FIG. 8C.

[230] For quantitative analysis of CanAg levels, CanAg serum levels were those reported for patients who participated in the cantuzumab-martansine clinical trial prior to treatment (Tolcher et al., J. Clin. Oncol. 21 (2). : 211-22 (2003)). A CanAg standard curve was generated using a CanAg standard using an ELISA assay similar to that described for DS6. C242 was used to capture the CanAg standard. Detection of captured CanAg was achieved using a biotinylated C242 tracer followed by development with streptavidin-HRP using ABTS as the substrate. As was done for biotinylated DS6, a biotinylated C242 standard curve was constructed to allow units / ml to be converted to molar concentrations of circulating CanAg epitopes. In Table 6, CanAg levels from cantuzumab-matansin clinical trial patients are reported along with the corresponding calculated molar concentrations of circulating CanAg.
Table 6

Circulating CanAg pretreatment levels measured by ¹ sandwich ELISA
^Two goat anti-mouse IgG and biotin-C242 standard curves
³ Biotin-C242 standard curve

  [231] Comparison of pM levels of shed CA6 in patients with ovarian cancer and those calculated for shed CanAg in patients with CanAg-positive cancer generally indicates that shed CA6 levels are similar to shed CanAg levels. Furthermore, only 2 out of 16 ovarian cancer patient serum samples potentially have CA6 levels higher than 4.5 nM (signals outside standard curve range, serum samples 5 and 9), this level Higher than this was the level at which altered Herceptin pharmacokinetics was observed in clinical trials in Her2 / neu positive breast cancer patients. CanAg levels above 4.5 nM were seen only in 3 of 37 clinical trial patients. In this clinical trial, there was no correlation between shedding CanAg levels and the faster clearance of cantuzumab martansine. However, patients with the highest CanAg level (31240 U / ml) were sampled only 8 hours after infusion. These results indicate that certain epitopes of Mucl, such as CA6 and CanAg, are lost in cancer patients, but not to levels that prohibit antibody therapy treatment.

Example 6: Cloning of murine DS6 antibody variable region [232] Murine monoclonal antibodies such as DS6 are recognized as foreign by the human immune system and thus have limited utility in clinical settings. Patients develop human anti-mouse antibodies (HAMA) rapidly, resulting in rapid clearance of murine antibodies. For this reason, the surface of the variable region of murine DS6 (muDS6) was reconstituted to generate humanized DS6 (huDS6) antibodies.

  [233] The murine DS6 antibody variable region was cloned by RT-PCR. Total RNA was purified from confluent T175 flasks of DS6 hybridoma cells using a Qiagen RNeasy miniprep kit. RNA concentrations were measured by UV spectrophotometry and RT reactions were performed with 4-5 μg total RNA using Gibco Superscript II kit and random hexamer primers.

[234] Wang Z et al., J Immunol Methods. Jan 13; 233 (1-2): A PCR reaction was performed using degenerate primers based on those described in 167-77 (2000). The RT reaction mixture was used directly for the degenerate PCR reaction. 3 'light chain primer, HindKL,
(TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC) (SEQ ID NO: 25)
And 3 ′ heavy chain primer, BamIgG1,
(GGAGGATCCATAGACAGATGGGGGGTGTCGTTTTGGC) (SEQ ID NO: 26)
And for the 5 ′ end, the PCR primer is Sac1MK for the light chain.
(GGGAGCTCGAYATTGTGMTSACMCARWCTMCA) (SEQ ID NO: 27)
And for the heavy chain, EcoR1MH1
It was an equivalent mixture of (CTTCCGGAATTCSSARGTNMGCTGSAGSAGTC) (SEQ ID NO: 28) and EcoR1MH2 (CTCTCCGGAATTCSARGTNMMAGCTGSAGSAGCTCGG) (SEQ ID NO: 29) (mixed bases: H = A + T + C, S + T, C = G + C, Y = C = A + G, W = A + T, V = A + C + G, N = A + T + G + C).

[235] PCR reactions were standard except supplemented with 10% DMSO (50 μl reaction mix was final concentration 1 × reaction buffer (ROCHE), 2 mM dNTP each, 1 mM primer each, 2 μl RT reaction, 5 μl DMSO, and 0.5 μl Taq (ROCHE)). 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 1); 4) 72 ° C., 2 minutes; 5) 29 cycles back to step 2; 6) 72 ° C., 10 minutes final extension at 10 min final extension step, MJ
The PCR reaction was performed on a research thermocycler. The PCR product generated by the PCR primers was cloned into pBluescript II SK + (Stratagene) using restriction enzymes. The heavy and light chain clones were sequenced by the Seqwright sequencing service.

[236] Further PCR and cloning were performed to confirm the 5 ′ end cDNA sequence. The DS6 light chain and heavy chain cDNA sequences determined from the degenerate PCR clones were typed into NCBI's Blast search website and the murine antibody sequences with the signal sequences presented were recorded. PCR primers were designed from these signal peptides using stretches conserved between related DNA sequences. EcoRI restriction sites were added to the leader sequence primers (Table 7) and these were used in the RT-PCR reaction as described above.

  [237] Several individual light and heavy chain clones were sequenced and identified, and sequence errors that could be generated by the polymerase were avoided. Only a single sequence was obtained for both light and heavy chain RT-PCR clones. These sequences were sufficient to design primers that could be expanded into the signal sequence to amplify murine DS6 light and heavy chain sequences. Subsequent clones from these additional PCR reactions confirmed the 5 'end sequence of the variable region modified by the original degenerate primer. Cumulative results from various cDNA clones provided the final murine DS6 light and heavy chain sequences shown in FIG. Using the Kabat and AbM definitions, three light and heavy chain CDRs were identified (Figures 9 and 10). By searching the NCBI IgBlast database, the muDS6 antibody light chain variable region is most likely derived from the murine IgVκ ap4 germline gene, while the heavy chain variable region can be derived from the murine IgVh J558.41 germline gene. It is shown that the sex is the highest (FIG. 11).

Example 7: Determination of DS6 Antibody Variable Region Surface Residues [238] The antibody surface reconstruction technique described by Pedersen et al. (1994) and Roguska et al. (1996) predicts surface residues of murine antibody variable sequences. It starts with that. A surface residue is defined as an amino acid that has at least 30% of its total surface area accessible to water molecules. Because there was no elucidated structure to find surface residues for muDS6, we aligned the 10 antibodies with the most homologous sequences in the 127 antibody structure file set (FIG. 12). For these aligned sequences, the solvent accessibility at each Kabat position was averaged (FIGS. 13A and B).

  [239] By comparing antibody subsets containing two identical residues flanked on either side, surface positions with an average accessibility of between 25% and 35% were analyzed for the second cycle. (Figures 13A and B). After the second cycle analysis, for muDS6 heavy chain, 21 predicted surface residues increased to 23, and Tyr3 and Lys23 were listed in the list of predicted residues with higher surface accessibility than 30%. Added. Most of our surface reconstituted antibodies used the Kabat definition of heavy chain CDR1, but for DS6, the AbM definition was inadvertently used during the calculation, so the heavy chain residue T28 is a Kabat definition. Was not defined as it could be defined as a framework surface residue. The number of light chain surface positions was reduced from 16 to 15 because the predicted surface accessibility of Ala80 was reduced from 30.5% to 27.8% in the second period analysis. Taken together, the muDS6 heavy and light chain variable sequences have 38 predicted surface accessible framework residues.

Example 8: Human antibody selection [240] The surface position of the murine DS6 variable region was compared to the corresponding position in the human antibody sequence in the Kabat database (Johnson G, Wu TT. Nucleic Acids Res. Jan 1; 29 ( 1): 205-6 (2001)). Surface residues were extracted and aligned from natural heavy and light chain human antibody pairs using antibody database management software SR (Searle, 1998). Special consideration was given to positions within 5 of the CDRs and the human antibody variable region surface with the most identical surface residues was selected to replace the murine DS6 antibody variable region surface residues.

Example 9: Chimeric and Humanized Antibody Expression Vector [241] Light and heavy chain pairing sequences were cloned into a single mammalian expression vector. The human variable sequence PCR primer created a restriction site, allowing the human signal sequence to be added to the pBluescript II cloning vector. The variable sequences may then be cloned into mammalian expression plasmids with EcoRI and BsiWI or HidIII and ApaI for the light or heavy chain, respectively (FIG. 14). The light chain variable sequence was cloned in-frame onto the human Ig kappa constant region and the heavy chain variable sequence was cloned into the human Ig gamma 1 constant region sequence. In the final expression plasmid, the human CMV promoter drives the expression of both light and heavy chain cDNA sequences.

Example 10: Identification of residues that can negatively affect DS6 activity [242] To date, the majority of humanizations identify residues that are close to CDRs as potentially problematic residues A molecular model of the subject antibody has been constructed. As the number of surface reconstituted antibodies to start work is increasing, historical experience is at least as effective as building models to predict problems, and for DS6, We did not build a molecular model. Instead, murine DS6 surface residues were compared to previously surface-reconstituted antibodies, and residues that were high risk residues that affected antibody binding ability were identified.

  [243] In both the elucidated antibody structures available and molecular models derived from previous humanization, a similar set of residues was repeatedly identified as being within 5 of the CDRs. Using this data, Table 1 provides murine DS6 residues that are likely to be close to the CDRs and are probably within 5cm. Many of these positions have also been altered in previous humanizations, but so far only heavy chain position 74 has caused loss of binding activity. In order to preserve the binding activity of the murine antibody, a murine residue was retained at this position in both huC242 and huB4. On the other hand, this same position was changed in humanized 6.2G5C6 to the corresponding human residue, but without loss of activity (6.2G5C6 is often referred to as anti-IGF1- R antibody). Although any residue in Table 1 may present a problem in humanized antibodies, heavy chain residue P73 is of particular concern from previous experience at this position.

Example 11 Selection of Most Homologous Human Surfaces [244] SR software was used to derive candidate human antibody surfaces for reconstructing the muDS6 surface from the Kabat antibody sequence database. This software provides an interface for searching only specific residue positions against the antibody database. In order to preserve the natural pair, both light chain and heavy chain surface residues were compared together. The most homologous human surfaces from the Kabat database were aligned in rank order of sequence identity. Table 2 shows the top three surfaces when paralleled by SR Kabat database software. The surfaces were then compared to identify which human surface required the least changes to the residues identified in Table 1. The anti-Rh (D) antibody, 28E4 (Boucher et al., 1997) requires a minimal number of surface residue changes (total 11), and only three of these residues remain a potential problem. Included in the list of groups. Since the 28E4 antibody provides the most homologous human surface, this is the best candidate to reconstitute the muDS6 surface.

Example 12: Construction of DNA sequence for humanized DS6 antibody [245] Eleven surface residue changes for DS6 were made using PCR mutagenesis. Murine DS6 variable region cDNA clones were subjected to PCR mutagenesis to construct a surface reconstructed human DS6 gene. As shown in Table 8 below, a humanized primer set was designed to create the amino acid changes required for surface reconstitution DS6.
Table 8

  [246] PCR reaction was standard except supplemented with 10% DMSO (50 μl reaction mixture was final concentration 1 × reaction buffer (ROCHE), 2 mM each dNTP, 1 mM primer each, 100 ng template, 5 μl DMSO, and 0.5 μl Taq (ROCHE)). The following programs: 1) 94 ° C, 1 minute; 2) 94 ° C, 15 seconds; 3) 55 ° C, 1 minute; 4) 72 ° C, 1 minute; 5) 29 cycles back to step 2; 6) 72 ° C The PCR reaction was performed on an MJ Research thermocycler with termination at the final extension step of 4 minutes. The PCR product was digested with the corresponding restriction enzymes and cloned into the pBluescript cloning vector. Clones were sequenced to confirm amino acid changes.

  [247] Changing heavy chain residue P73 caused problems in the past, so we constructed two types of heavy chain, those with human 28E4 T73, and those that retain murine P73. In both forms of humanized DS6, the other 10 surface residues were changed from murine to human 28E4 residues (Table 2). The most human is the 1.0 type because it has all 11 human surface residues, following the usual nomenclature. For cases where additional types are needed, the heavy chain type that retains murine P73 is named 1.2, so type 1.1 is taken for the type containing the largest number of murine residues. deep. In FIGS. 15A and B, the two humanized amino acid sequences are shown aligned with the murine DS6 amino acid sequence. Both humanized DS6 antibody genes were cloned into antibody expression plasmids (FIG. 14) for transient and stable transfection. The cDNA and amino acid sequences of the humanized 1.01 and 1.21 light chain variable regions are the same and are shown in FIG. The heavy chain cDNA and amino acid sequences of humanized versions 1.01 and 1.21 are shown in FIGS.

Example 13: Expression and purification of huDS6 in CHO cells and affinity measurement [248] Expression and purification of antibodies to determine whether the humanized DS6 type retains the binding affinity of muDS6 Was necessary. CHO cells were stably transfected with a chimeric form of DS6 (chDS6) with human constant region and murine variable region, or with huDS6v1.01 or huDS6v1.21.

[249] CHODG44 cells (4.32 × 10 ⁶ cells / plate) were added to non-selective medium (alpha MEM + nucleotides (4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin, and 10% v / v FBS). Gibco)) were planted in 15 cm plates and placed in a 37 ° C., 5% CO ₂ humidified incubator. The next day, cells were transfected with the chDS6 expression plasmid using a modified version of the protocol recommended by Qiagen for Polyfect Transfection. Non-selective medium was aspirated from the cells. Plates were washed with 7 ml pre-warmed (37 ° C.) PBS and refilled with 20 ml non-selective medium. Plasmid DNA (11 μg) was diluted in 800 μl of hybridoma SFM (Gibco). 70 μl of Polyfect (Qiagen) was then added to the DNA / SFM mixture. The Polyfect mixture was then gently vortexed for a few seconds and incubated at ambient temperature for 10 minutes. Non-selective medium (2.7 ml) was added to the mixture. This final mixture was incubated with the plated cells for 24 hours.

[250] The transfection mixture / medium was removed from the plate and then the cells were trypsinized and counted. The cells were then placed in selective media (4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin, in 96-well plates (250 μl / well) at various densities (1800, 600, 200, and 67 cells / well). Plated in alpha MEM-nucleotide (Gibco) supplemented with 10% v / v FBS, 1.25 mg / ml G418. Cells were incubated for 2-3 weeks, supplemented with media if necessary. Wells were screened for antibody production levels using a quantitative ELISA. Immulon 2HB 96-well plates were coated with goat anti-human IgG F (ab) ₂ antibody (Jackson Immunoresearch; 1 μg / well in 100 μl 50 mM sodium carbonate buffer pH 9.6) and shaken at ambient temperature 1 Incubated for 5 hours. All subsequent steps were performed at ambient temperature. Wells were washed twice with T-TBS (0.1% Tween-20, TBS) and blocked for 1 hour with 200 μl blocking buffer (1% BSA, T-TBS). Wells were washed twice with T-TBS. In separate plates, antibody standards, EM164 (100 ng / ml) and culture supernatant were serially diluted (1: 2 or 1: 3) in blocking buffer. These dilutions (100 μl) were transferred to ELISA plates and incubated for 1 hour. Wells were washed 3 times with T-TBS and incubated for 45 minutes with 100 μl goat anti-human IgG Fc-AP (Jackson ImmunoResearch) diluted 1: 3000 in blocking buffer. After washing 5 times with T-TBS, 100 μl of PNPP coloring reagent (10 mg / ml PNPP (p-nitrophenyl phosphate, disodium salt; Pierce), 0.1 M diethanolamine pH 10.3 buffer) Was allowed to develop for 25 minutes. Absorbance at 405 nm was measured in an ELISA plate reader. Absorbance readings (of the culture supernatant) at the linear portion of the standard curve were used to determine antibody levels.

[251] The largest producing clone identified by ELISA was then subcloned, expanded, and a frozen cell stock prepared.
[252] For expression of huDS6v1.01 and huDS6v1.21, DG44 CHO cells (Dr. Lawrence Chasin, Columbia University, NY) were treated with alpha MEM (Gibco catalog number 12571, Grand Island, NY) containing ribonucleotides and deoxyribonucleotides. Incubated in. 10% fetal bovine serum (HyClone catalog number SH30071.03, Logan, UT), 1% gentamicin (Mediatech catalog number 30-005-CR, Herndon, VA), and 2 mM L-glutamine (L-glut) (BioWhittaker) Catalog number 17-605E, Walkersville, MD). This formulation was named CHO complete medium.

[253] DG44 CHO cells (5 × 10 ⁶ ) were transfected with 50 μg of huDS6 plasmid DNA. Prior to transfection, cells were removed from flasks with trypsin (Gibco catalog number 15090-046, Grand Island, NY) and lacking ribonucleosides and deoxyribonucleosides, non-supplemented alpha MEM (Gibco catalog number 12561, Grand Island, NY) And washed twice. This was named the washing medium. Cells were mixed with plasmid DNA in a 0.4 cm gap electrode cuvette (BioRad catalog number 1652088, Hercules, Pa.). These were placed on ice for 2 minutes and then pulsed at 1,000 μF and 260 volts in a BioRad electroporation apparatus. After electroporation, the cells were incubated for 2 minutes on ice. Cells were then plated in 5 24-well plates (Costar catalog number 3524) in CHO complete medium and maintained in a 37 ° C. incubator with 5% CO ₂ . After 48 hours, the medium was removed from the wells. Wells were rinsed once with wash medium and 1% gentamicin, 2 mM L-glut, 10% dialyzed fetal calf serum (Gibco catalog number 26400-044, Grand Island, NY), and 1.25 mg / ml Geneticin (G418) Alpha MEM (Gibco catalog number 12561, Grand Island, NY) without ribonucleosides and deoxyribonucleosides supplemented (Gibco catalog number 11811, Grand Island, NY) was supplied. This complete formulation was named selective medium. Clones were incubated for approximately 2 weeks in selective medium, at which point they were screened for antibody production by quantitative ELISA. The highest producing clone was then subcloned, expanded, and a frozen cell stock prepared.

[254] In order to produce sufficient amount of antibody to purify, on 15cm plates (~1x10 ⁶ cells / plate) containing selective medium 30ml supplemented with Ultra Low IgG FBS (Gibco), to expand the cells and 1 Incubated for a week. Culture supernatant was collected in a 250 ml conical tube, spun down (2000 rpm, 5 min, 4 ° C.) in a tabletop centrifuge and then sterile filtered through a 0.2 μm filter device.

[255] To purify DS6, a NaOH pellet was added to the filtered culture supernatant to a final pH of 8.0. A HiTrap r Protein A column (Amersham) was equilibrated with 20-50 column volumes of binding buffer. The supernatant was loaded onto the column using a peristaltic pump. The column was then washed with 50 column volumes of binding buffer. The elution buffer (100 mM acetic acid, 50 mM NaCl, pH 3) was used to elute the bound antibody from the column into a test tube set in the fraction collector. Neutralized buffer (2M K ₂ HPO ₄ , pH 10.0) was used to neutralize the eluted antibody and dialyzed overnight in PBS. The dialyzed antibody was filtered through a 0.2 μm syringe filter. Absorbance at 280 nm was measured to determine the final protein concentration.

[256] The affinity of purified huIgG was compared to muDS6 by flow cytometry. In the first experimental set, direct binding to the CA6-expressing cell line, KB, was measured. As shown in FIG. 18, muDS6, chDS6, huDS5v1.01 and huDS6v1.21 show very similar affinity, and the apparent Kd is 0.82 nM, 0.69 nM, 0.82 and 0. 0, respectively. 85 nM, suggesting that surface reconstruction did not interfere with the CDRs. To confirm that huDS6 type retains muDS6 affinity, competitive binding experiments were performed. The advantage of this format is that it uses the same detection system for both murine and human antibodies; ie biotin-muDS6 / streptavidin-DTAF. The results of a competitive binding assay comparing the ability of muDS6, chDS6, huDS6v1.01 and huDS6v1.21 to compete with biotin-DS6 is shown in FIG. The apparent EC ₅₀ is 1.9 nM, 1.7 nM, 3.0 and 1.9 nM for muDS6, chDS6, huDS6v1.01 and huDS6v1.21, respectively. These results indicate that muDS6 surface reconstitution to produce humanized DS6 causes little loss of binding affinity.

Example 14: Preparation of muDS6-DM1 cytotoxic conjugate [257] An 8 fold molar excess of N-succinimidyl-4- (2-pyridyldithio) pentanoate (SPP) was used to generate muDS6 antibody (8 mg / ml). Modified to introduce a dithiopyridyl group. The reaction was performed in 95% v / v buffer 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 gel filtered through a NAP or Sephadex G25 column (equilibrated in buffer A). The degree of modification was determined by measuring antibody absorbance at 280 nm and 2-mercaptopyridine (Spy) released at DTT at 280 and 343 nm. The modified muDS6 was then 2.5 mg Ab using a 1.7-fold molar excess of N ^{2 ′} -deacetyl-N ^{2 ′} -(3-mercapto-1-oxopropyl) -maytansine (L-DM1) over SPy. Conjugated at / ml. The reaction was performed in buffer A (97% v / v) containing DMA (3% v / v). The reaction was incubated overnight at room temperature for ˜20 hours. The opaque reaction mixture was centrifuged (1162 × g, 10 minutes), and then NAP-25 or S300 (Tandem 3, 3 × 26/10 desalting column, G25 medium equilibrated in buffer B (1 × PBS pH 6.5) ) The supernatant was gel filtered through the column. The pellet was discarded. The conjugate was sterile filtered using a 0.22 μm Millex-GV filter and dialyzed in Buffer B using a Slide-A-Lizer. The number of DM1 molecules linked per molecule of muDS6 was determined by measuring the absorbance of both the 252 nm and 280 nm of the filtered material. The DM1 / Ab ratio was found to be 4.36 and the process yield of conjugated MUDS6 was 55%. The conjugated antibody concentration was 1.32 mg / ml. The purified conjugate was biochemically characterized by size exclusion chromatography (SEC) and found to be 92% monomer. Analysis of DM1 in the purified conjugate showed that 99% was covalently bound to the antibody. In FIG. 20, flow cytometry binding of muDS6-DM1 conjugate and unmodified muDS6 to Caov-3 cells shows that conjugation of muDS6 results in only a slight loss of affinity.

Example 15: In vitro cytotoxicity of muDS6-DM1 [258] As a naked antibody, muDS6 showed no proliferation or growth inhibitory activity in cell culture (FIG. 21). However, when muDS6 is incubated with cells in the presence of DM1 conjugates for goat anti-mouse IgG heavy and light chains, muDS6 is very effective at targeting and delivering this conjugate to cells; Indirect cytotoxicity occurred (Figure 21). To further test the innate activity of naked muDS6, a complement dependent cytotoxicity (CDC) assay using muDS6 was performed. 5% human or rabbit serum and various dilutions in 200 μl RHBP medium (RPMI-1640, 0.1% BSA, 20 mM HEPES (pH 7.2-7.4), 100 U / ml penicillin and 100 μg / ml streptomycin) HPAC and ZR-75-1 cells (25000 cells / well) were plated in 96 well plates in the presence of muDS6. Cells were incubated for 2 hours at 37 ° C. Then Ala
Mar Blue (final concentration 10%) reagent (Biosource) was added to the supernatant. Cells were incubated for 5-24 hours before measuring fluorescence. Murine DS6 had no effect in the complement dependent cytotoxicity (CDC) assay (FIG. 22). This suggests that therapeutic application of muDS6 may require conjugation of toxic effector molecules.

[259] The cytotoxicity of maytansinoid-conjugated muDS6 antibodies was examined in a variety of DS6-positive cell lines using two different assay formats. A colony formation assay was performed in which cells (1000-2500 cells / well) were plated on 6-well plates in 2 ml conjugate diluted in medium. Generally, cells are continuously exposed to the conjugate at several concentrations between 3 × 10 ⁻¹¹ M and 3 × 10 ⁻⁹ M and in a 37 ° C., 6% CO ₂ humidified chamber for 5-9 days. Incubated. Wells were washed with PBS and colonies were stained with 1% w / v crystal violet / 10% v / v formaldehyde / PBS solution. The unbound stain was then thoroughly washed from the wells using double distilled water and the plates were allowed to dry. Leica
Colonies were counted using a StereoZoom 4 dissecting microscope.

[260] The plating efficiency (PE) was calculated as number of colonies / number of cells plated. Viability was calculated as PE of treated cells / PE of untreated cells. IC ₅₀ concentration was determined by graphing cell viability versus molar concentration of conjugate. In the colony formation assay (FIG. 23), muDS6-DM1 was effective in killing Caov-3 cells with an estimated IC ₅₀ of 800 pM. The antigen negative cell, A375, was only slightly affected by the 3 × 10 ⁻⁹ M concentration conjugate, which is the highest concentration of muDS6-DM1 tested, and the cell killing activity of the conjugate was reduced to antigen expressing cells. Proved to be specifically directed. However, despite the apparent sensitivity to maytansine, many other DS6 positive cell lines were not particularly sensitive to immunoconjugates. All cervical cell lines (HeLa, KB, and WISH) were sensitive to the conjugate, while only a selected number of ovarian and breast cell lines showed some cytotoxic effect. None of the pancreatic cell lines appeared to be affected.

[261] Cells were seeded in 96-well plates at a density of 1000-5000 cells / well in the MTT assay. Cells were plated with serial dilutions of either naked muDS6 or muDS6-DM1 immunoconjugate in 200 μl medium. Samples were run in triplicate. The cells and antibody / conjugate mixture were then incubated for 2-7 days, at which point by MTT ([3 (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide]) assay. Cell viability was assessed. MTT (50 μg / well) was added to the culture supernatant and allowed to incubate at 37 ° C. for 3-4 hours. The medium was removed and MTT formazan was solubilized in DMSO (175 μl / well). Absorbance at 540-545 nm was measured. In the MTT cell viability assay (FIG. 24C), the immunoconjugate was able to effectively kill Caov-3 cells with an estimated IC ₅₀ of 1.61 nM. Compared to naked antibodies that had no effect at all, the wells containing the highest concentration of conjugate did not contain any viable cells (FIGS. 21 and 24).

  [262] The results of the MTT assay for other cell lines were slightly different (Figures 24A, B, and DI). In many cases, some cytotoxicity was seen, but the conjugate was not able to completely kill the entire cell population (except for WISH cells). BT-20, OVCAR5, and HPAC cells were particularly resistant: in wells with the highest conjugate concentration (32 nM), more than 50% of the cells were still viable.

Example 16: In vivo conjugate antitumor activity [263] Human tumor xenografts were established in SCID mice to demonstrate the in vivo activity of muDS6-DM1 conjugate. A subcutaneous model of the human cervical cancer cell line KB was developed. KB cells were grown in vitro, harvested, and injected under the right shoulder of each mouse with 5 × 10 ⁶ cells in 100 μl of serum-free medium and grown for 6 days to an average tumor volume of 144 ± 125 mm ³ At this point, drug treatment was started. Mice were treated with PBS, 150 μg / kg DM1 conjugate, or 225 μg / kg.
Either of the DM1 conjugates (2 mice per group) was administered intravenously daily for 5 days. During the treatment, the toxic response was monitored daily. Throughout the study, tumor volume (FIG. 25A) and corresponding body weight (FIG. 25B) were monitored.

  [264] KB tumors treated with PBS control grew rapidly with a doubling time of about 4 days. In contrast, both groups of mice treated with the conjugate show complete tumor regression in the 225 μg / kg and 150 μg / kg dose groups, respectively, 14 and 18 days after the start of treatment. At the 150 μg / kg dose, the tumor delay was approximately 70 days. Treatment with 225 μg / kg resulted in healing when there was no evidence of tumor recurrence when the study was terminated on day 120. As seen in FIG. 25B, mice in the 150 μg / kg group did not show any weight loss, indicating that the dose was well tolerated. At higher doses, mice experience only a temporary 3% decrease in body weight. During the 5-day course of treatment, the mice showed no visible signs of toxicity. Taken together, this study demonstrates that muDS6-DM1 treatment can cure mice with KB xenograft tumors at non-toxic doses.

[265] MuDS6-DM1 activity was further tested against a panel of subcutaneous xenograft models (see FIG. 26). The tumor cell lines used to generate the xenografts showed various in vitro maytansine sensitivities and CA6 epitope density (Table 9 below). OVCAR5 cells and TOV-21G are ovarian tumor cell lines; HPAC is a pancreatic tumor cell line; HeLa is a cervical tumor cell line. OVCAR5 and TOV-21G cells have low surface CA6 expression; HeLa cells have intermediate levels of surface CA6 expression; HPAC cells have high CA6 surface expression density. TOV-21G and HPAC cells were maytansine sensitive; OVCAR5 and HeLa cells were 2-7 times less sensitive to maytansine.
Table 9

^* Average maximum relative average fluorescence

[266] Four cell lines were grown and collected in vitro, and 1 × 10 ⁷ cells in 100 μl of serum-free medium were injected under the right shoulder of each mouse (6 mice per model) and In the OVCAR5 test group and the control group, 57.6 ± 6.7 and 90.2 ± 13.4 mm ³ , respectively, and in the HPAC test group and the control group, 147.1 ± 29.6 and 176.2 ± 18, respectively. .9 mm ³ , 194.3 ± 37.2 and 201.7 ± 71.7 mm ^{3 in} the HeLa test group and the control group, respectively, and 96.6 ± 22. ^{3 in} the TOV-21G test group and the control group, respectively. Drug treatment was initiated at this point for 6 days to grow to an average tumor volume of 8 and 155.6 ± 13.4 mm ³ . 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 intravenous conjugate (600 μg / kg DM1). During the treatment, toxic responses were monitored daily and tumor volume and body weight were monitored throughout the study. The conjugate effectiveness of the various models is shown graphically in FIGS. 26A, C, E, and G, and the corresponding body weights are plotted in FIGS. 26B, D, F, and H. OVCAR5, TOV-21G, and HPAC cell lines form aggressive tumors as can be seen in the PBS control for each model. The HeLa model had a delay period of about 3 weeks before beginning exponential growth. In all models, DS6-DM1 conjugate treatment resulted in complete tumor regression in all mice. For the TOV-21G, HPAC, and HeLa models, mice remain tumor free on day 61. In the OVCAR5 model, tumors recurred approximately 45 days after tumor inoculation. Thus, in this model, muDS6-DM1 treatment results in a tumor growth delay of approximately 34 days. Growth delay is significant because OVCAR5 cells are less maytansine sensitive and have low CA6 epitope expression. Tumor regression is more robust in models where the CA6 epitope density is higher or the model has higher maytansine sensitivity. It is important to note that only two doses are administered. Because no weight loss is observed, the dosing schedule used in this study is clearly not toxic to mice. Healing may be achievable with additional or higher conjugate doses.

[267] Human ovarian cancer is mostly a peritoneal disease. OVCAR5 cells proliferate aggressively as an intraperitoneal (IP) model in SCID mice, forming tumor nodes and producing ascites in a manner similar to human disease. To prove activity in the IP model,
MuDS6-DM1 was used to treat mice bearing OVCAR5 IP tumors (FIG. 27). OVCAR5 cells were grown in vitro, harvested and injected intraperitoneally with 1 × 10 ⁷ cells in 100 μl of serum-free medium. Tumors were allowed to grow for 6 days, at which point treatment began. Mice were treated weekly for 2 weeks with either PBS or DS6-DM1 conjugate at a dose of 600 μg / kg DM1, and monitored for weight loss resulting from peritoneal disease. By day 28, the PBS group of mice lost more than 20% and was euthanized. After losing more than 20% body weight, the treatment group was sacrificed on day 45. This study shows that muDS6-DM1 can delay tumor growth in an aggressive OVCAR5 IP model, despite the fact that OVCAR5 cells are less sensitive to maytansine and have fewer CA6 epitopes per cell. It is proved that there is. Because the dosing schedule used did not induce visible signs of toxicity, it is likely that additional doses and higher doses could be used to achieve further tumor growth delay or healing.

Example 17: Synthesis and Characterization of DS6-SPP-MM1-202 Taxoid Cytotoxic Conjugate [268] muDS6 with N-sulfosuccinimidyl 4-nitro-2-pyridyl-pentanoate (SSNPP) linker Was modified. To 50 mg muDS6 Ab in 90% Buffer A, 10% DMA, 10 equivalents of SSNPP in DMA was added. The final concentration of Ab was 8 mg / ml. The reaction was stirred at room temperature for 4 hours and then purified by G25 chromatography. The absorbance of 280 nm (antibody) and 325 (linker) was used to measure the degree of antibody modification spectrophotometrically and was found to have 3.82 linker / antibody. Antibody recovery was 43.3 mg, yielding an 87% yield. muDS6-nitro SPP was conjugated with taxoid MM1-202 (1812P.16). Conjugation was performed on a 42 mg scale in 90% buffer A, 10% DM1. The taxoid was added at 4 aliquots of 0.43 equivalents / linker (each aliquot) over a period of about 20 hours. By this point, the reaction was significantly cloudy. The resulting conjugate, recovered in about 64% yield after G25 purification, had about 4.3 taxoid / Ab and left about 1 equivalent of unreacted linker. To stop the reaction of the unreacted linker, 1 equivalent of cysteine / unreacted linker was added to the conjugate with stirring overnight. When cysteine was added, a distinct yellowish hue was prominent, indicating the release of thiopyridine. The reaction solution was then dialyzed in buffer B / 0.01% Tween 20 and then dialyzed for several more days with buffer B alone. The final conjugate had 2.86 drugs / antibody. Antibody recovery was 14.7 mg, yielding a total yield of 35%. The conjugate was further biochemically characterized by SEC and was found to have 89% monomer, 10.5% dimer and 0.5% higher molecular weight aggregates.

  [269] The results of flow cytometry analysis comparing the binding of muDS6-SPP-MM1-202 taxoid vs. muDS6 antibody to HeLa cells are shown in FIG. The results indicate that muDS6 retains binding activity when conjugated to a taxane.

Example 18: In vitro and in vivo activity of humanized DS6 conjugate [270] huDS6v1.01-SPDB-DM4 conjugate was constructed. This conjugate is similar to the muDS6-SPP-DM1 conjugate described in Example 14 except that the structure of the linker / maytansine drug moiety of the conjugate differs around the disulfide bond; the muDS6-SPP-DM1 conjugate The gate has one methyl group steric hindrance on the disulfide carbon on the antibody side of the linker, while the SPDB-DM4 conjugate has two methyl group steric hindrance on the disulfide carbon on the maytansine side of the linker.

[271] The huDS6v1.01 antibody (8 mg / ml) was modified with an 8-fold molar excess of N-succinimidyl-4- (2-pyridyldithio) butanoate (SPDB) to introduce a dithiopyridyl group. The reaction was performed in 95% v / v buffer A (50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v / v ethanol at room temperature for 1.5 hours. The reaction mixture was gel filtered through a 15 ml Sephadex G25 column (equilibrated in buffer A). The degree of modification was determined by measuring antibody absorbance at 280 nm and 2-mercaptopyridine (Spy) released at DTT at 280 and 343 nm. The modified DS6 was then used with a 1.7-fold molar excess of N ^{2 ′} -deacetyl-N ^{2 ′} -(4-methyl-4-mercapto-1-oxopentyl) -maytansine (L-DM4) over SPy. Conjugated with 1.8 mg Ab / ml. The reaction was performed in buffer A (97% v / v) containing DMA (3% v / v). The reaction was incubated overnight at room temperature for ˜20 hours. In contrast to the conjugation of muDS6, the reaction mixture is clear and immediately gel filtered through a NAP 15 ml G25 column equilibrated in citrate buffer (20 mM citrate, 135 mM NaCl, pH 5.5). It went through. The conjugate was sterile filtered using a 0.22 μm Millex-GV filter. The number of DM4 molecules linked per molecule of DS6 was determined by measuring absorbance at both 252 nm and 280 nm of the filtered material. The DM4 / Ab ratio was found to be 3.2 and the process yield of conjugated DS6 was 69%. The conjugated antibody concentration was 1.51 mg / ml. The purified conjugate was biochemically characterized by size exclusion chromatography (SEC) and found to be 92.5% monomer. Analysis of DM4 in the purified conjugate showed> 99% covalently bound to the antibody.

[272] FIG. 29A shows that huDS6v1.01 conjugation essentially does not result in loss of affinity due to flow cytometric binding of huDS6v1.01-DM4 conjugate and unmodified DS6 to KB cells. . Binding was performed essentially as described for FIG. 20, except that KB cells were used rather than CaOv-3 cells. In vitro cytotoxicity of huDS6v1.01 was tested essentially as described in FIG. 24G. huDS6v1.01 killed WISH cells with an IC ₅₀ of 4.4 × ^{10 −10} M, whereas unconjugated huDS6v1.01 showed no cytotoxic activity.

[273] The in vivo activity of huDS6v1.01-DM4 was tested against the HPAC pancreas model. HPAC cells were inoculated on day 0 and immunoconjugate treatment was administered on day 13. PBS control animals were euthanized when the tumor volume exceeded 1000 cm ³ . The conjugate was administered at a dose of either 200 μg / kg or 600 μg / kg DM4, corresponding to antibody concentrations of 15 mg / kg and 45 mg / kg, respectively. During the course of the study, mice were monitored for tumor volume (Figure 30A) and body weight (Figure 30B). huDS6v1.01-DM4 showed potent anti-tumor activity at 200 μg / kg DM4 and all mice achieved complete tumor regression. A control B4-DM4 conjugate that recognizes an antigen not expressed on HPAC xenografts was essentially inactive at 200 μg / kg. The lack of weight loss in mice (FIG. 30B) indicates that treatment with the 200 μg / kg conjugate is below the maximum tolerated dose. This result demonstrates that the humanized form of DS6 can mediate targeted delivery of maytansinoid drugs and produce potent antitumor activity.

[33] FIG. 1 shows the results of studies conducted to determine the ability of DS6 antibody to bind to the surface of selected cancer cell lines. The fluorescence of cell lines incubated with DS6 primary antibody and FITC conjugated anti-mouse IgG (H + L) secondary antibody was measured by flow cytometry. The DS6 antibody bound to Caov-3 (FIG. 1A) and T-47D (FIG. 1B) cells, and the apparent Kd was 1.848 nM and 2.586 nM, respectively. Antigen negative cell lines, SK-OV-3 (FIG. 1C) and Colo205 (FIG. 1D) did not show any antigen-specific binding. [34] FIG. 2 shows the results of a dot blot analysis of epitope expression. Caov-3 (FIGS. 2A and 2B), SKMEL28 (FIG. 2C), and Colo205 (FIG. 2D) cell lysates were individually spotted onto nitrocellulose membranes and then pronase, proteinase K, neuraminidase or periodate Individual incubation with acid. The membrane was then immunoblotted with DS6 antibody (FIG. 2A), CM1 antibody (FIG. 2B), R24 antibody (FIG. 2C), or C242 antibody (FIG. 2D). [35] FIG. 3 shows the results of dot blot analysis of DS6 antigen expression. Caov-3 cell lysates were individually spotted on PVDF membranes and then incubated in the presence of trifluoromethanesulfonic acid (TFMSA). The membrane was then immunoblotted with CM1 antibody (1 and 2) or DS6 (3 and 4). [36] FIG. 4 shows the results of glycotope analysis of the DS6 antigen. Caov-3 lysate pretreated with N-glycanase (“N-gly”), O-glycanase (“O-gly”), and / or sialidase (“S”) is spotted onto nitrocellulose, and Subsequently, immunoblotting was performed with DS-6 antibody or CM1 antibody (Muc-1 VNTR). [37] FIG. 5 shows the results of Western blot analysis of the DS6 antigen. Cell lysates were immunoprecipitated (“IP”) with DS6 antibody and immunoblotted. The antigen corresponds to the> 250 kDa protein band observed in antigen positive Caov-3 (FIGS. 5A and 5B) and T47D (FIG. 5C) cells. Antigen negative SK-OV-3 (FIG. 5D) and Colo205 (FIG. 5E) cell lines do not show this band. Following immunoprecipitation, Caov-3 cell lysate protein G beads were incubated with (FIG. 5A) neuraminidase (“N”) or (FIG. 5B) periodic acid (“PA”). Antibody (“α”), pre-IP (“Lys”) and post-IP flow-through (“FT”) lysate controls were run on the same gel. Caov-3 immunoprecipitates are also incubated with N-glycanase (“N-gly”), O-glycanase (“O-gly”), and / or sialidase (“S”) (see FIG. 5F). ), In this case, the blot was instead probed with biotinylated DS6 and streptavidin-HRP. [37] FIG. 5 shows the results of Western blot analysis of the DS6 antigen. Cell lysates were immunoprecipitated (“IP”) with DS6 antibody and immunoblotted. The antigen corresponds to the> 250 kDa protein band observed in antigen positive Caov-3 (FIGS. 5A and 5B) and T47D (FIG. 5C) cells. Antigen negative SK-OV-3 (FIG. 5D) and Colo205 (FIG. 5E) cell lines do not show this band. Following immunoprecipitation, Caov-3 cell lysate protein G beads were incubated with (FIG. 5A) neuraminidase (“N”) or (FIG. 5B) periodic acid (“PA”). Antibody (“α”), pre-IP (“Lys”) and post-IP flow-through (“FT”) lysate controls were run on the same gel. Caov-3 immunoprecipitates are also incubated with N-glycanase (“N-gly”), O-glycanase (“O-gly”), and / or sialidase (“S”) (see FIG. 5F). ), In this case, the blot was instead probed with biotinylated DS6 and streptavidin-HRP. [38] FIG. 6 shows the results of immunoprecipitation and / or immunoblotting of DS6 and CM1 antibodies against Caov-3 (FIG. 6A) and HeLa (FIG. 6B) cell lysates. The overlap of CM1 and DS6 Western blot signals indicates that the DS6 antigen is on the Mucl protein. In HeLa lysates, Mucl doublets arise from Mucl expression expressed by distinct alleles that differ in the number of tandem repeats. [39] Figure 7 shows the DS6 antibody sandwich ELISA design (Figure 7A) and standard curve (Figure 7B). A standard curve was generated using known concentrations of the commercially available CA15-3 standard (1 CA15-3 units = 1 DS6 units). [40] FIG. 8 shows a quantitative ELISA standard curve. Using a known concentration of biotin-DS6, either captured by plated goat anti-mouse IgG (FIG. 8A) or directly bound on an ELISA plate (FIG. 8B), a detection antibody ( A standard curve of streptavidin-HRP / biotin-DS6) signal (FIG. 8C) was determined. [41] FIG. 9 shows the cDNA and amino acid sequences of the light chain (FIG. 9A) and heavy chain (FIG. 9B) variable regions of murine DS6 antibody. The three CDRs in each sequence are underlined (Kabat definition). [42] FIG. 10 shows the light chain (FIG. 10A) and heavy chain (FIG. 10B) CDRs of murine DS6 antibody as determined by the Kabat definition. AbM modeling software gives a slightly different definition of heavy chain CDRs (FIG. 10C). [43] FIG. 11 shows the murine DS6 antibody light chain (“muDS6LC”) (residue of SEQ ID NO: 7), juxtaposed with the germline sequences of the IgVκap4 (SEQ ID NO: 23) and IgVh J588.41 (SEQ ID NO: 24) genes. Groups 1-95) and heavy chain ("muDS6HC") (residues 1-98 of SEQ ID NO: 9) amino acid sequences. Gray indicates sequence differences. [44] FIG. 12 shows the 10 light and heavy chains most homologous to the murine DS6 (muDS6) light chain (“muDS6LC”) and heavy chain (“muDS6HC”) sequences with resolved structure files in the Brookhaven database. The chain antibody sequence is shown. Sequences are juxtaposed in order from the most homologous to the least homologous. [45] FIG. 13 shows surface accessibility data and calculations that predict which framework residues of the murine DS6 antibody light chain variable region are accessible to the surface. The place with an average surface accessibility of 25-35% was marked ( ^* ?? ^* ) and was subjected to the second cycle analysis. DS6 antibody light chain variable region (FIG. 13A) and heavy chain variable region (FIG. 13B). 〓 [45] FIG. 13 shows surface accessibility data and calculations that predict which framework residues of the murine DS6 antibody light chain variable region are accessible to the surface. The place with an average surface accessibility of 25-35% was marked ( ^* ?? ^* ) and was subjected to the second cycle analysis. DS6 antibody light chain variable region (FIG. 13A) and heavy chain variable region (FIG. 13B). [46] FIG. 14 shows the prDS6 v1-0 mammalian expression plasmid map. This plasmid was used to construct and express recombinant chimeric and humanized DS6 antibodies. [47] FIG. 15 shows the amino acid sequence of murine (“muDS6”) and humanized (“huDS6”) (1.01 and 1.21) DS6 antibody light chain (FIG. 15A) and heavy chain (FIG. 15B) variable domains. Indicates. [48] FIG. 16 shows the cDNA and amino acid sequences of the light chain variable region of the humanized DS6 antibody (“huDS6”) (1.01 and 1.21). [49] FIG. 17 shows the cDNA and amino acid sequences of the heavy chain variable region of humanized DS6 antibody (“huDS6”) 1.01 (FIG. 17A) and 1.21 (FIG. 17B). [50] FIG. 18 shows murine DS6 (muDS6), chimeric DS6 (chDS6), and human DS6 1.01 type (huDS6 v1.01) and huDS6 1.21 type from assays performed on KB cells ( 2 shows a flow cytometry binding curve of huDS6 v1.21). The avidity of murine, chimeric, and human v1.01 and v1.21 DS6 antibodies (muDS6 = 0.82 nM, chDS6 = 0.69 nM, huDS6v1.01 = 0.82 nM and huDS6v1.21 = 0.85 nM) are comparable Indicating that surface reconstruction did not reduce avidity. [51] FIG. 19 shows the results of a competitive binding assay of muDS6, chDS6, huDS6 v1.01 and huDS6 v1.21 antibodies and biotinylated muDS6. Various concentrations of naked muDS6, chDS6, huDS6v1.01 and huDS6v1.21 were combined with 2 nM biotin-muDS6 and streptavidin-DTAF secondary antibody. All antibodies have similar IC50s (muDS6 = 1.9 nM, chDS6 = 1.7 nM, huDS6v1.01 = 3.0 nM, and huDS6v1.21 = 1.9 nM), which shows that humanization reduces avidity Indicates that it was not. [52] FIG. 20 shows the results of binding affinity determination of unconjugated DS6 antibody to DS6 antibody-DM1 conjugate. This result indicated that DM1 conjugation did not adversely affect the binding affinity of the antibody. The apparent Kd (3.902 nM) (“DS6-DM1”) of the DS6 antibody-DM1 conjugate was slightly higher than the naked antibody (2.020 nM) (“DS6”). [53] FIG. 21 shows the results of an indirect cell viability determination assay using DS6 antibody in the presence or absence of anti-mouse IgG (H + L) DM1 conjugate (2 ° Ab-DM1). Antigen positive Caov-3 cells were killed in a DS6 antibody dependent manner (IC ₅₀ = 424.9 pM) only in the presence of the secondary conjugate (“DS6 + 2 ° Ab-DM1”). [54] FIG. 22 shows the results of the complement dependent cytotoxicity (CDC) assay of muDS6 antibody. The results indicate that CDC-mediated DS6 antibody has no effect on HPAC (FIG. 22A) and ZR-75-1 (FIG. 22B) cells. [55] Figure 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate against free maytansine. In a colony formation assay, DS6 antigen-positive ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C), and pancreatic cancer (FIG. 23D) cell lines were serially exposed to DS6 antibody-DM1 conjugate. Tested for cytotoxicity (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours of exposure to unbound maytansine (right panel). 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. [55] Figure 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate against free maytansine. In a colony formation assay, DS6 antigen-positive ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C), and pancreatic cancer (FIG. 23D) cell lines were serially exposed to DS6 antibody-DM1 conjugate. Tested for cytotoxicity (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours of exposure to unbound maytansine (right panel). 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. [55] Figure 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate against free maytansine. In a colony formation assay, DS6 antigen-positive ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C), and pancreatic cancer (FIG. 23D) cell lines were serially exposed to DS6 antibody-DM1 conjugate. Tested for cytotoxicity (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours of exposure to unbound maytansine (right panel). 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. [55] Figure 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate against free maytansine. In a colony formation assay, DS6 antigen-positive ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C), and pancreatic cancer (FIG. 23D) cell lines were serially exposed to DS6 antibody-DM1 conjugate. Tested for cytotoxicity (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours of exposure to unbound maytansine (right panel). 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. [56] FIG. 24 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate. In the MTT cell viability assay, human ovarian cancer (Figures 24A, 24B and 24C), breast cancer (Figures 24D and 24E), cervical cancer (Figures 24F and 24G), and pancreatic cancer (Figures 24H and 24I). ) Cells were killed in a DS6 antibody-DM1 conjugate dependent manner. Naked DS6 did not adversely affect the growth of these cells, indicating that cytotoxicity requires DM1 conjugation. [57] FIG. 25A shows the results of an in vivo anti-tumor efficacy study of DS6 antibody-DM1 conjugates against established subcutaneous KB tumor xenografts. Tumor cells were inoculated on day 0 and the first treatment was given on day 6. Immunoconjugate treatment was continued daily for a total of 5 doses. PBS control animals were euthanized when the tumor volume exceeded 1500 mm ³ . The conjugate was administered at a dose of 150 or 225 μg / kg DM1, which corresponded to antibody concentrations of 5.7 and 8.5 mg / kg, respectively. During the course of the study, the body weight of the mice (FIG. 25B) was monitored. [58] FIG. 26 shows the results of an anti-tumor efficacy study of DS6 antibody-DM1 conjugates against established subcutaneous tumor xenografts. On day 0, OVCAR5 (Figures 26A and 26B), TOV-21G (Figures 26C and 26D), HPAC (Figures 26E and 26F), and HeLa (Figures 26G and 26H) cells are inoculated and immune Conjugate treatment was given on days 6 and 13. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of 600 μg / kg DM1, corresponding to an antibody concentration of 27.7 mg / kg. During the course of the study, mouse tumor volumes (FIGS. 26A, 26C, 26E, and 26G) and body weights (FIGS. 26B, 26D, 26F, and 26H) were monitored. [58] FIG. 26 shows the results of an anti-tumor efficacy study of DS6 antibody-DM1 conjugates against established subcutaneous tumor xenografts. On day 0, OVCAR5 (Figures 26A and 26B), TOV-21G (Figures 26C and 26D), HPAC (Figures 26E and 26F), and HeLa (Figures 26G and 26H) cells are inoculated and immune Conjugate treatment was given on days 6 and 13. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of 600 μg / kg DM1, corresponding to an antibody concentration of 27.7 mg / kg. During the course of the study, mouse tumor volumes (FIGS. 26A, 26C, 26E, and 26G) and body weights (FIGS. 26B, 26D, 26F, and 26H) were monitored. [58] FIG. 26 shows the results of an anti-tumor efficacy study of DS6 antibody-DM1 conjugates against established subcutaneous tumor xenografts. On day 0, OVCAR5 (Figures 26A and 26B), TOV-21G (Figures 26C and 26D), HPAC (Figures 26E and 26F), and HeLa (Figures 26G and 26H) cells are inoculated and immune Conjugate treatment was given on days 6 and 13. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of 600 μg / kg DM1, corresponding to an antibody concentration of 27.7 mg / kg. During the course of the study, mouse tumor volumes (FIGS. 26A, 26C, 26E, and 26G) and body weights (FIGS. 26B, 26D, 26F, and 26H) were monitored. [58] FIG. 26 shows the results of an anti-tumor efficacy study of DS6 antibody-DM1 conjugates against established subcutaneous tumor xenografts. On day 0, OVCAR5 (Figures 26A and 26B), TOV-21G (Figures 26C and 26D), HPAC (Figures 26E and 26F), and HeLa (Figures 26G and 26H) cells are inoculated and immune Conjugate treatment was given on days 6 and 13. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of 600 μg / kg DM1, corresponding to an antibody concentration of 27.7 mg / kg. During the course of the study, mouse tumor volumes (FIGS. 26A, 26C, 26E, and 26G) and body weights (FIGS. 26B, 26D, 26F, and 26H) were monitored. [59] FIG. 27 shows the results of an in vivo efficacy study of muDS6 antibody-DM1 conjugates against intraperitoneal OVCAR5 tumors. On day 0, tumor cells were injected intraperitoneally and on days 6 and 13, immunoconjugate treatment was given. Animals were euthanized when weight loss exceeded 20%. [60] FIG. 28 shows a flow cytometry binding curve from a binding affinity study of naked and taxane-conjugated DS6 antibodies to HeLa cells. Taxane (MM1-202) -conjugation does not adversely affect the binding affinity of the antibody. The apparent Kd (1.24 nM) of the DS6-MM1-202 conjugate was slightly larger than the naked DS6 antibody (620 pM). [61] FIG. 29 shows the in vitro binding and strength of humanized DS6 type 1.01 antibody conjugates. Conjugation of DM4 and huDS6v1.01 has little effect on the avidity of huDS6v1.01 on KB cells (FIG. 29A). huDS6v1.01-DM4 showed potent in vitro cytotoxicity towards WISH cells expressing DS6 with an IC ₅₀ of 0.44 nM (FIG. 29B). [62] FIG. 30 shows the results of an in vivo efficacy study using huDS6v1.01-DM4 conjugate in an HPAC pancreatic cancer model. huDS6v1.01-DM4 showed potent anti-tumor activity, whereas the B4-DM4 control conjugate, in which no target was expressed in the HPAC model, had essentially no activity (FIG. 30A). The administered dose of 200 μg / kg was not toxic to the animals as indicated by the lack of weight loss (FIG. 30B).

Claims (20)

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, wherein the heavy chain variable region or fragment thereof is SEQ ID NO: 9, sequence An amino acid sequence selected from the group consisting of No. 10 and SEQ ID No. 11:
Wherein said antibody or epitope-binding fragment thereof has at least 90% sequence identity.   2. The antibody or epitope-binding fragment thereof of claim 1, wherein the heavy chain variable region or fragment thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.   The antibody or epitope-binding fragment thereof according to claim 1, wherein the heavy chain variable region or a fragment thereof has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. The light chain variable region or a fragment thereof is an amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8:
The antibody or epitope-binding fragment thereof of claim 1, having at least 90% sequence identity to the antibody. The antibody or epitope-binding fragment thereof of claim 1, wherein the light chain variable region or fragment thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.   The antibody or epitope-binding fragment thereof according to claim 1, wherein the light chain variable region or a fragment thereof has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.   The heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, and the light chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. The antibody or epitope-binding fragment thereof according to claim 1, wherein   A polynucleotide encoding the antibody or epitope-binding fragment thereof according to claim 1.   A polynucleotide encoding the antibody or epitope-binding fragment thereof according to claim 4.   A polynucleotide encoding the antibody or epitope-binding fragment thereof according to claim 7.   A polynucleotide encoding the heavy chain of the antibody or epitope-binding fragment thereof according to claim 1.   An expression vector comprising the polynucleotide of claim 8.   An expression vector comprising the polynucleotide of claim 9.   An expression vector comprising the polynucleotide of claim 10.   A host cell comprising the expression vector of claim 12.   A host cell comprising the expression vector of claim 13.   A host cell comprising the expression vector of claim 14. A method for preparing an antibody or epitope-binding fragment thereof, comprising culturing the host cell of claim 15 under conditions that promote expression of said antibody or epitope-binding fragment thereof, and said polypeptide from said cell culture. Wherein the antibody or 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 heavy chain variable region or fragment thereof is An amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11:
Wherein said method comprises a step having at least 90% sequence identity. A method for preparing an antibody or epitope-binding fragment thereof, comprising culturing the host cell of claim 16 under conditions that promote expression of said antibody or epitope-binding fragment thereof, and said polypeptide from said cell culture. Wherein the heavy chain variable region or fragment thereof is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11:
An amino acid sequence having at least 90% sequence identity and wherein the light chain variable region or fragment thereof is represented by SEQ ID NO: 7 or SEQ ID NO: 8:
Wherein said method comprises a step having at least 90% sequence identity. A method for preparing an antibody or epitope-binding fragment thereof, comprising culturing the host cell of claim 17 under conditions that promote expression of said antibody or epitope-binding fragment thereof, and said polypeptide from said cell culture. Wherein the heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, and the light chain variable region or fragment thereof is SEQ ID NO: 7 or The method comprising the step of having the amino acid sequence of SEQ ID NO: 8.