HK1125030A - Rs7 antibodies - Google Patents

Description

RS7 antibody

The present application is a divisional application of the patent application having application number 03809918.7 entitled "RS 7 antibody".

1. Field of the invention

The present invention relates to monovalent and multivalent, monospecific binding proteins and to multivalent, multispecific binding proteins. One embodiment of these binding proteins contains one or more binding sites, wherein each binding site binds to a target antigen or an epitope on a target antigen. Another embodiment of these binding proteins contains two or more binding sites, wherein each binding site has affinity for a different epitope on the target antigen or has affinity for the target antigen or hapten. The invention also relates to recombinant vectors for expressing these functional binding proteins in a host. More specifically, the invention relates to a tumor associated antigen binding protein designated RS 7. The invention also relates to humanized RS7 antigen binding proteins, and the use of the binding proteins in diagnosis and therapy.

2. Background of the invention

Artificial binding proteins, particularly monoclonal antibodies and engineered treatment antibodies or antibody fragments, have been extensively tested and shown to be of value in the detection and treatment of a variety of human diseases, including cancer, autoimmune diseases, infectious diseases, inflammatory diseases and cardiovascular diseases (Filpula and McGuire, exp. Opin. Ther. patents (1999) 9: 231-. For example, radioisotope-labeled antibodies have been detected for use in visualizing tumors following injection into a patient using art-available detection agents. The clinical utility of an antibody or antibody-derived agent depends largely on its ability to bind to a specific target antigen. The selectivity capability is useful for delivering a diagnostic or therapeutic agent, such as an isotope, drug, toxin, cytokine, hormone, growth factor, enzyme, conjugate, radionuclide or metal, to a target site during the detection and treatment of human disease, particularly if the diagnostic or therapeutic agent is toxic to normal tissues in the body.

Goldenberg, The American Journal of Medicine (1993) 94: 298-299 discloses potential limitations of the antibody system. Important parameters in detection and therapeutic techniques are the amount and uptake rate of the injected dose, particularly the dose at the site of presence of the target cells, i.e. the ratio of the concentration of specifically bound antibody to the radioactivity present in the surrounding normal tissue. When an antibody is injected into the bloodstream, it is metabolized and excreted through multiple compartments. Antibodies must be able to localize and bind to target cell antigens as they pass through other parts of the body. Factors controlling antigen targeting include location, size, antigen density, antigen accessibility, cellular composition of pathological tissues and pharmacokinetics of the targeting antibody. Other factors that can particularly affect the targeting of antibodies to tumors include the expression of the target antigen in tumors and other tissues and bone marrow toxicity caused by the slow blood clearance rate of radiolabeled antibodies. The amount of targeting antibody attached to the targeted tumor cells is affected by vascularization and penetration of the tumor antibody through the barrier, as well as intratumoral pressure. Non-specific uptake by non-target organs such as liver, kidney or bone marrow is another potential limitation of this technique, particularly for use in transmission immunotherapy, where irradiation of bone marrow often results in dose-limiting toxicity.

One proposed approach, direct targeting, is a technique designed to target antigens with antibodies carrying diagnostic or therapeutic radioisotopes. In the field of tumors, direct targeting methods use radiolabeled anti-tumor monospecific antibodies that recognize tumors by their antigens. The technique involves injecting a labeled monospecific antibody into a patient and then allowing the antibody to localize to the target tumor to obtain a diagnostic or therapeutic effect. Unbound antibody is cleared by the body. The method can be used to diagnose or treat additional mammalian diseases.

Another proposed solution, referred to as "affinity enhancement System" (AES), (U.S. Pat. No. 5,256,395(1993), Barbet et al, Cancer Biotherapy & Radiopharmaceuticals (1999) 14: 153- "166) designed specifically to overcome the deficiencies of tumor targeting with antibodies carrying diagnostic or therapeutic radioisotopes. AES employs radiolabeled hapten and an anti-tumor/anti-hapten bispecific binding protein that recognizes the target tumor and the radioactive hapten. Haptens with higher valency and binding proteins with higher specificity can also be used in this method. The method comprises injecting the binding protein into a patient and then localizing it to the target tumor. After a sufficient period of time for the unbound binding protein to be cleared from the bloodstream, a radiolabeled hapten is administered. The hapten binds to the antibody-antigen complex at the target cellular site to achieve a diagnostic or therapeutic effect. Unbound hapten is cleared by the body. Barbet describes the possibility that a bivalent hapten can be cross-linked with a bispecific antibody when the latter is bound to the tumor surface. As a result, the radiolabeled complex is more stable and remains in the tumor for a longer period of time. The system can be used for diagnosing or treating a disease in a mammal.

There remains a need in the art for the preparation of multivalent, monospecific binding proteins that can be used in direct targeting systems and for the preparation of multivalent, multispecific binding proteins that can be used in affinity enhancement systems. In particular, there remains a need for binding proteins that exhibit enhanced uptake of targeted antigens, reduced blood levels, and optimal protection of normal tissues and cells from toxic drugs.

Summary of The Invention

Accordingly, it is an object of the present invention to provide monospecific monoclonal antibodies and fragments thereof recognizing tumor associated antigens, epithelial glycoprotein-1 (EGP-1) as defined by murine MAb RS7-3G11 against human non-small cell lung cancer. The RS7 antigen has been named EGP-1 (epithelial glycoprotein-1) according to the third International Association for the study of Lung cancer (IASLC) recommendations for tumor and differentiation antigens. At least one epitope associated with EGP-1 is also referred to in the literature as TROP 2. In a preferred embodiment, the antibody or antibody fragment of the invention binds to the same epitope as Stein (below) and other previously described murine RS7 antibodies. Alternatively, the epitope bound by the antibody or fragment may be different from the epitope bound by the murine RS7 antibody disclosed by Stein. In a preferred embodiment, the anti-EGP-1 or anti-TROP 2 antibody or fragment thereof is a chimeric, humanized or fully human RS7 antibody or fragment thereof.

For example, the invention relates to a humanized antibody or fragment thereof, wherein the Complementarity Determining Regions (CDRs) of the light chain variable region of the humanized RS7MAb comprise CDR1 comprising the amino acid sequence KASQDVSIAVA; a CDR2 comprising an amino acid sequence of SASYRYT; and CDR3 comprising the amino acid sequence QQHYITPLT. Another embodiment of the invention is a humanized antibody or fragment thereof, wherein the CDRs of the heavy chain variable region of the humanized RS7MAb comprise CDR1 comprising the amino acid sequence NYGMN; a CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG and a CDR3 comprising the amino acid sequence GGFGSSYWYFDV. Also preferably, the humanized antibody or fragment thereof comprises the CDRs of the murine RS7MAb and the Framework Regions (FRs) of the light and heavy chain variable regions of the human antibody, wherein the CDRs of the light chain variable region of the humanized RS7MAb comprise CDR1 comprising the KASQDVSIAVA amino acid sequence; a CDR2 comprising an amino acid sequence of SASYRYT; and CDR3 comprising the amino acid sequence QQHYITPLT; and the CDRs of the heavy chain variable region of the humanized RS7MAb comprise CDR1 comprising the amino acid sequence NYGMN; a CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG and a CDR3 comprising the amino acid sequence GGFGSSYWYFDV. Also preferably, the humanized antibody or fragment thereof further comprises the FRs of the light and heavy chain constant regions of a human antibody.

In a preferred embodiment, the humanized RS7 antibody or fragment comprises FRs of the light and/or heavy chain comprising at least one amino acid substituted by an amino acid residue found at a corresponding position in the murine RS7 antibody. For example, wherein at least one of the substituted amino acid residues is preferably at a position selected from the group consisting of amino acid residues 38, 46, 68 and 91 of the murine heavy chain variable region of FIG. 3B and/or at least one of the substituted amino acid residues is preferably at a position selected from the group consisting of amino acid residues 20, 85 and 100 of the murine light chain variable region of FIG. 3A.

The invention also features an antibody fusion protein or fragment thereof comprising at least two anti-EGP-1 MAbs or fragments thereof, wherein the MAbs or fragments thereof are selected from the anti-EGP-1 MAbs or fragments thereof of the invention. In a related vein, an antibody fusion protein or fragment thereof includes at least one first anti-EGP-1 MAb or fragment thereof of any anti-EGP-1 antibody of the present invention and at least one second MAb or fragment thereof other than an anti-EGP antibody of the present invention. For example, the second antibody or fragment thereof can be a cancer-associated antibody or fragment thereof. Another preferred embodiment is a fusion protein or fragment thereof comprising two different epitope-binding anti-EGP-1 antibodies or fragments thereof.

It is an object of the present invention to provide multispecific antibodies and fragments thereof that recognize more than one epitope on the RS7 antigen or that have affinity for the RS7 antigen and for hapten molecules. The latter binding protein is suitable for pre-targeting a target antigen. Thus, also described is a method of delivering a diagnostic agent, a therapeutic agent, or a combination thereof to a target comprising: (i) administering to the individual a multivalent, multispecific MAb or fragment thereof; (ii) waiting a sufficient amount of time to allow the amount of non-binding protein to be cleared from the blood stream of the individual; and (iii) administering to the individual a diagnostic agent, a therapeutic agent, or a combination thereof that binds to the binding site of the individual.

It is another object of the invention to provide methods for delivering diagnostic or therapeutic agents to targeted diseases expressing EGP-1 antigens. For example, a method of delivering a diagnostic agent or a therapeutic agent, or a combination thereof, to a target is described, comprising (i) providing a composition comprising an anti-EGP-1 antibody or fragment thereof that binds to at least one therapeutic and/or diagnostic agent and (ii) administering the composition to the individual in need thereof. Preferably, the diagnostic or therapeutic agent is selected from the group consisting of isotopes, drugs, toxins, immunomodulators, hormones, enzymes, growth factors, radionuclides, metals, contrast agents and detection agents.

In another embodiment of the invention, a method of delivering a diagnostic agent, a therapeutic agent, or a combination thereof to a target site comprises (i) administering to an individual a multivalent, multispecific antibody or fragment comprising one or more antigen-binding sites having affinity for a target antigen of EGP-1 and one or more hapten binding sites having affinity for a hapten molecule; (ii) waiting a sufficient amount of time to allow the amount of non-binding protein to be cleared from the blood stream of the individual; and (iii) administering to the individual a composition comprising a diagnostic agent, a therapeutic agent, or a combination thereof.

It is another object of the present invention to provide a diagnostic or therapeutic conjugate of a cancer cell-targeted antibody fusion protein or fragment thereof comprising an anti-EGP-1 MAb or fragment thereof or any one of the antibodies of the present invention wherein the anti-EGP-1 antibody or fragment thereof is conjugated to at least one diagnostic or therapeutic agent. Suitable therapeutic agents are drugs having a pharmacological property selected from the group consisting of antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic, alkaloid and antibiotic agents, and combinations thereof.

Also preferred are therapeutic agents selected from the group consisting of nitrogen mustards, aziridine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, tyrosine kinase inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, antagonists, endostatin, taxols, camptothecins, doxorubicin analogs, and combinations thereof. Preferably, the diagnostic agent is selected from the group consisting of a photoactivatable radionuclide, preferably 25-4000 keV, and a contrast agent.

In a preferred embodiment, the DNA sequence comprises a nucleic acid encoding an anti-EGP-1 MAb or fragment thereof of the invention; an antibody fusion protein or fragment thereof comprising at least two of said MA b or fragments thereof; an antibody fusion protein or fragment thereof comprising at least one first anti-EGP-1 MAb or fragment thereof comprising the MAb or fragment thereof of the anti-EGP-1 antibodies and fragments of the invention and at least one second MAb or fragment thereof other than the anti-EGP-1 MAb or fragment thereof described herein; or an antibody fusion protein or fragment thereof comprising at least one first MAb or fragment thereof comprising the MAb or fragment thereof of any antibody described herein and at least one second MAb or fragment thereof other than the MAb or fragment thereof of any antibody described herein, wherein the second MAb is reactive with an antigen selected from the group consisting of EGP-2, MUC1-4, a33, CSAp, CEA, Le (y), Tn, Tag-72, PSMA, PSA, EGFR, HER2/neu, AFP, HCG- β, ferritin, PAP, PLAP, EGP-2, histone, cytokeratin, tenascin, CanAg, kidney cancer G250, VGFR1, VGFR2, PAM 4-antigen, oncogene products, and combinations thereof. The second Mab may alternatively be reactive with tumor-associated vascular endothelial antigens, such as VEGF (vascular endothelial growth factor) and P1GF (placental growth factor). The choice of the second antibody depends on the tumor cell type. For example, anti-PSMA or anti-PSA antibodies may be used in the treatment or diagnosis of prostate cancer, anti-CEA or anti-MUCL, MUC2, MUC3 and MUC4 antibodies for breast, ovarian, lung and colon cancer, EGFR for colon and head and neck cancer, anti-CSAP antibodies for colon and ovarian cancer, and anti-HER/neu for breast, ovarian and other cancers. These are for illustrative purposes only and are not intended to be limiting. Expression vectors and host cells containing the DNA sequences are also preferred embodiments of the invention.

Also provided herein are methods of diagnosing and treating malignancies. For example, a method of diagnosing and treating cancer, comprising (i) administering to an individual in need thereof a multivalent, multispecific antibody or fragment comprising one or more antigen-binding sites having affinity for an EGP-1 target antigen and one or more hapten binding sites having affinity for a hapten molecule; (ii) waiting a sufficient amount of time to allow the amount of non-binding protein to be cleared from the blood stream of the individual; and (iii) administering to the individual a hapten comprising a diagnostic agent, a therapeutic agent, or a combination thereof that binds to the binding site of the antibody.

Likewise, methods of diagnosing and treating malignancies may comprise administering a therapeutically effective amount of an anti-EGP-1 fusion protein or fragment thereof or a therapeutic conjugate comprising an EGP-1MAb or fragment thereof, wherein the EGP-1MAb or fragment thereof or antibody fusion protein or fragment thereof is bound to at least one therapeutic agent in a pharmaceutically suitable excipient. In the relevant vein, naked anti-EGP-1 antibodies and fragments thereof, including naked anti-EGP-1 fusion proteins and fragments thereof, may also be used to treat malignancies. Naked anti-EGP-1 antibodies can be used for in vitro diagnosis of malignancies, for example using immunoassay or immunohistochemistry, but cannot be used for in vivo diagnosis unless they include pre-targeting techniques such as AES. However, labeled EGP-1 antibodies are useful for in vivo diagnosis and treatment of malignancies. For example, described herein are methods of treating cancer cells in an individual comprising (i) administering to the individual a therapeutically effective amount of a composition comprising an anti-EGP-1 MAb or fragment thereof or an antibody fusion protein or fragment thereof, (ii) formulating the EGP-1MAb or fragment thereof or antibody fusion protein or fragment thereof in a pharmaceutically suitable excipient. Similarly, the present invention also includes combinations of naked mabs and fragments thereof and conjugated mabs or fragments thereof or fusion proteins or fragments thereof for use in diagnosis and therapy.

Brief Description of Drawings

FIG. 1 shows a comparison of mRS7, cAb-Vkappa #23(cRS7), and CAB-Vkappa #1 in a competitive binding assay. Different concentrations of competing Ab were used to compete with the binding of biotinylated mRS7 antibody constant. The results showed that vk #1 light chain did not bind RS7 antigen.

FIG. 2 shows the DNA and amino acid sequences encoding (A) RS7V κ cloned by 5' RACE and (B) RS7VH cloned by RT-PCR. The putative CDR regions are underlined. Nucleotide residues are numbered consecutively. Kabat's Ig molecular numbering was used for amino acid residues. In (B), the numbering of residues by letter (top) is by the number of the preceding residue plus the letter, e.g., the number of T after N52 is 52A; the numbers of N, N and L after 182 are 82A, 82B and 82C, respectively.

FIG. 3 shows (A) human SA-1A' c1, murine RS7, and hRS7V_κChain sum (B) human RF-TS3, murine RS7, and hRS7V_HAlignment of the amino acid sequences of the chains. In (A), the dots indicate the same residues in RS7 as the corresponding residues in SA-1A' c 1. The dashed lines indicate gaps introduced to aid alignment. Boxes represent CDR regions. Both the N-and C-terminal residues of hRS7 (underlined) were immobilized by the stagging vector used. Thus, the corresponding terminal residue of RS7 cannot be compared to that in the human sequence. Kabat's numbering scheme was used. In (B), the dots show the same residues in RS7 as the corresponding residues in RF-TS 3. The dashed lines indicate gaps introduced to aid alignment. Boxes represent CDR regions. Both the N-and C-terminal residues of hRS7 (underlined) were immobilized by the stagging vector used. Thus, the corresponding terminal residue of RS7 cannot be compared to that in the human VH sequence.

FIG. 4 shows (A) humanized RS7V κ and (B) humanized RS7V_HThe DNA and amino acid sequence of (1). The bold and underlined portions of the amino acid sequences represent the CDRs as determined using the Kabat's numbering scheme.

FIG. 5 shows (A) the light chain cDNA and amino acid sequence of humanized RS7V kappa and humanized RS7V_HHeavy chain cDNA and amino acid sequence of (a). The underlined part of the amino acid sequence indicates the leader peptide sequence for secretion. "" denotes a stop codon.

FIG. 6 shows a comparison of mRS7, cRS7 and hRS7 in a competitive binding assay. Different concentrations of competitor Ab were used to compete with biotinylated RS7 antibody constant for binding to Ag-coated 96-well ELISA plates. hRS7 showed comparable blocking activity to RS7 and cRS 7.

Figure 7 shows the light chain cDNA and amino acid sequence of humanized RS7V κ. The underlined part of the amino acid sequence indicates the leader peptide sequence for secretion. "" denotes a stop codon. Lysine residues are also underlined.

Figure 8 shows the heavy chain cDNA and amino acid sequence of humanized RS7V κ. The underlined part of the amino acid sequence indicates the leader peptide sequence for secretion. "" denotes a stop codon. Lysine residues are also underlined.

FIG. 9 shows the structures of the remaining portions IMP-R4, IMP-R5 and IMP-R8.

FIG. 10 is a bar graph of dose determination of hRS7 in the MDA-MB-468 tumor model due to radioiodination.

Figure 11 provides a set of graphs demonstrating the effect of radioimmunotherapy on tumor growth of breast cancer xenografts in nude mice.

FIG. 12 is a set of graphs evaluating toxicity following radioimmunotherapy of breast cancer xenografts in nude mice.

Fig. 13 is a graph showing relative Mean Tumor Volume (MTV).

Detailed description of the preferred embodiments

The terms "a" or "an" mean "one or more" unless specified otherwise.

The RS7 antibody (formerly known as RS7-3G11) is a murine IgG produced against a crude membrane preparation of human primary squamous cell carcinoma of the lung₁. See Stein et al, Cancer res.50: 1330(1990), the entire disclosure of which is incorporated by reference. The RS7 antibody recognizes a tumor-associated antigen, as determined by the murine MAb RS7-3G11, raised against human non-cell lung cancer. Stein et al disclose an RS7 antibody recognizing the 46-48kDa glycoprotein, characterized by cluster 13. Stein et al, int.j.cancer supp.8: 98-102(1994). See also, Basu et al, int.j. cancer 52: 472-479(1995). According to 3^rdThe International Association for the study of Lung cancer (IASLC) has proposed a tumor and differentiation antigen, which has been designated EGP-1 (epithelial glycoprotein-1). See, e.g., DeLeij et al, int.j. cancer supp., 8: 60-63(1994). Thus, RS7 and EGP-1 antigen have the same meaning as described herein. EGP-1 antigen also refers to TROP2 in the prior art, but may be a multiple epitope of both EGP-1 and TROP 2.

Flow cytometry and immunohistochemical staining studies have shown that RS7Mab can detect antigens on a variety of tumor types, while it only binds to normal human tissues to a limited extent. (Stein et al, (1990), supra). The RS7 antibody reacts with EGP-1 glycoprotein which can be rapidly internalized. EGP-1 is expressed primarily by cancers such as lung, stomach, bladder, breast, ovary, uterus and prostate cancer. Localization and therapeutic studies using radiolabeled murine RS7Mab in animal models have demonstrated the efficacy of tumor targeting and therapy (Stein et al, (1990), supra.

More recent studies have demonstrated strong RS7 staining in tumors from lung, breast, bladder, ovary, uterus, stomach and prostate. See Stein et al, int.j. cancer 55: (938) (1993), the entire contents of which are incorporated herein by reference. Moreover, the lung cancer cases in this study were squamous cell carcinoma and adenocarcinoma. As before. Both cell types stained strongly, indicating that the RS7 antibody cannot be distinguished from the histological classification of non-small cell lung cancer.

As described above, RS7 mabs were rapidly internalized into target cells (Stein et al (1993), supra). The internalization rate constant of the RS7MAb is between that of the other two rapidly internalizing mabs that have been shown to be useful for immunotoxin preparation. As before. There have been numerous reports indicating that internalization of immunotoxin conjugates is absolutely essential for anti-tumor activity. (PASTAN et al, Cell 47.641 (1986)). Internalization of drug immunoconjugates has also been described as a major factor in anti-tumor efficacy. (Yang et al, Proc. Nat' l. Acad. Sci. USA 85: 1189 (1988)). Thus, the RS7 antigen may be an important target for these types of immunotherapy that require internalization of the therapeutic agent.

Thus, studies with RS7Mab have shown that antibodies exhibit certain important properties that make them candidates for clinical diagnostic and therapeutic applications. Because the RS7 antigen provides a useful target for diagnosis and therapy, it is desirable to obtain mabs that recognize epitopes of the RS7 antigen. Moreover, the effectiveness of chimeric, humanized and human RS7 antibodies is fundamental to the development of a dual determinant enzyme-linked immunosorbent assay (ELISA), which requires detection of the RS7 antigen in two samples and is fundamental to human in vivo applications.

To this end, the present invention describes chimeric, humanized and human antibodies and fragments thereof that bind to RS7 antigen and are useful in diagnostic and therapeutic methods. Humanized antibodies and antibody fragments have been described in U.S. provisional application No. 18733/1073, U.S. provisional application No. 60/356,132, U.S. provisional application No. 60/416,232, and human surrogate No. 18733/1155, entitled "anti-CD 20 antibodies and fusion proteins thereof and methods of use"; hMN-14 antibodies, such as those disclosed in U.S. application No. 5,874,540, which are class III anti-carcinoembryonic antigen antibodies (anti-CEA antibodies); mu-9 antibodies, such as those disclosed in U.S. application No. 10/116,116; AFP antibodies, such as those disclosed in U.S. provisional application 60/399,707; PAM4 antibodies, such as those disclosed in U.S. provisional application entitled "monoclonal antibody cPAM4," attorney docket No. 18733/1102; RS7 antibodies, such as those disclosed in U.S. provisional application 60/360,229; and CD22 antibodies, such as those disclosed in U.S. patent nos. 5,789,554 and 6,187,287 and U.S. application nos. 09/741,843 and 09/988,013, all of which are incorporated herein by reference in their entirety. The chimeric antibody described herein is a recombinant protein comprising variable regions comprising Complementarity Determining Regions (CDRs) derived from an antibody of one species, preferably a rodent antibody, while the constant regions of the antibody molecule are derived from a human antibody. For veterinary applications, the constant region of the chimeric antibody may be derived from other species. A humanized antibody is a recombinant protein in which the CDRs of an antibody from a species, such as a rodent antibody, are transferred from the heavy and variable light chains of the rodent antibody to the human heavy and light chain variable regions.

In a preferred embodiment, the RS7 antibody is humanized. Because non-human monoclonal antibodies can be recognized by the human host as foreign proteins and repeated injections can result in harmful allergic reactions, humanization of the murine RS7 sequence can reduce the adverse immune response experienced by the patient. For mice basedThe monoclonal antibody of (a), which is generally referred to as a human anti-mouse antibody (HAMA) response. Another embodiment of the invention is an anti-EGF-1 antibody or fragment thereof, which is a less than human primate anti-EGP-1 antibody, murine monoclonal anti-EGP-1 antibody (limited to veterinary applications), chimeric anti-EGP-1 antibody, human anti-EGP-1 antibody, and humanized anti-EGP-1 antibody. Preferably, certain human residues in the framework regions of the humanized RS7 antibody or fragment thereof are replaced with murine analogues. Also preferably, a combination of framework sequences from two different human antibodies is used for V_H. The constant regions of the antibody molecule are derived from those of human antibodies.

Another preferred embodiment of the invention is a human RS7 antibody. Human antibodies are antibodies obtained from transgenic mice that have been "engineered" to make specific human antibodies in response to antigenic challenge. In this method, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines containing targeted disruptions of the endogenous heavy and light chain loci. The transgenic mouse can synthesize human antibody with specificity aiming at human antigen, and the mouse can be used for preparing hybridoma secreting human antibody. Green et al Nature Genet.7: 13(1994), Lonberg et al Nature 368: 856(1994) and Taylor et al int.Immun.6: 579(1994) describes a method for obtaining human antibodies from transgenic mice. Fully human antibodies can also be constructed using gene or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See, for example, McCafferty et al, Nature 348: 552-553(1990) human antibodies and fragments thereof were prepared in vitro from immunoglobulin variable region gene libraries from non-immunized donors. In this method, antibody variable region genes are cloned in-frame into the major or minor outer membrane protein genes of filamentous phage and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particle comprises a single DNA copy of the phage genome, selection based on the functional properties of the antibody will also result in selection of genes encoding antibodies exhibiting these properties. In this approach, the phage mimics certain characteristics of the B cell. Phage display can be performed in a variety of formats, for a review of which see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571(1993).

The antibodies and fragments thereof of the present invention are preferably produced against crude membrane preparations from human primary squamous cell lung cancer. Also preferably, the RS7 antibodies and fragments thereof are produced against membrane preparations of live cells from human ovarian cancer cell lines. Also preferably, the RS7 antigen is provided by a live Colo 316 cell. In the relevant vein, RS7 antibodies can be obtained using substantially pure preparations of RS7 antigen. A substantially pure protein is a protein that is substantially free of contaminating cellular components with which the protein is associated in its native state. As used herein, the term "RS 7 antibody" also includes chimeric, human and humanized RS7 antibodies.

Preparation of chimeric, humanized and human RS7 antibodies

Monoclonal antibodies directed against specific antigens can be obtained using methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256: 495(1975), and Coligan et al (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, Vol.1, pp.2.5.1-2.6.7 (John Wiley & Sons 1991) (hereinafter "Coligan"). Briefly, RS7 antigen mabs, such as RS7, can be prepared by injecting mice with a composition comprising RS7 antigen, identifying the presence of antibody preparation by taking a serum sample, obtaining B-lymphocytes from the spleen, fusing the B-lymphocytes with myeloma cells to prepare hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to RS7 antigen, culturing the clones that produce antibodies to RS7 antigen, and isolating RS7 antibody from the hybridoma culture.

After initial production of antibodies to the antigen, the antibodies can be sequenced and subsequently prepared using recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. For example, humanized monoclonal antibodies are prepared by transferring mouse complementarity determining regions from heavy and light variable chains of a mouse immunoglobulin into human variable regions, and then replacing human residues in the framework regions with murine analogs. The use of antibody components derived from humanized monoclonal antibodies avoids potential problems associated with murine constant region immunogenicity.

The human antibodies of the invention, i.e., human EGP-1MAb or other human antibodies, such as anti-EGP-2, MUC1-4, CEA, CC49, CSAP, PSMA, PSA, EGFR, A33, and HER2/neu MAb, useful for combination therapy with humanized, chimeric, or human RS7 antibodies, can be obtained from transgenic non-human animals. See, e.g., Mendez et al, Nature Genetics, 15: 146-156 (1997); U.S. patent 5,633,425, which is incorporated herein by reference in its entirety. The human antibodies of the invention useful in combination therapy may also react with an antigen selected from le (y), Tn, Tag-72, AFP, HCG-beta, ferritin, PAP, EGP-2, histone, cytokeratin, tenascin, CanAg, renal carcinoma G250, VGFR1, VGFR2, or combinations thereof. For example, human antibodies can be recovered from transgenic mice containing human immunoglobulin loci. The mouse humoral immune system is humanized by inactivation of endogenous immunoglobulin genes and introduction of human immunoglobulin loci. The human immunoglobulin locus is very complex and contains a large number of discrete segments, which together account for almost 0.2% of the human genome. To ensure that transgenic mice are able to produce an adequate pool of antibodies, most of the human heavy and light chain loci must be introduced into the mouse genome. This is accomplished by a stepwise approach, beginning with the construction of Yeast Artificial Chromosomes (YACs) containing immunoglobulin loci in germline configuration, either human heavy or light chains. Since the size of the uninserted insert is about 1Mb, homologous recombination of immunoglobulin locus repeats is required for YAC construction. Two YACs, one containing the heavy chain locus and one containing the light chain locus, were introduced separately into mice via fusions containing YAC-yeast spheroblasts with mouse embryonic stem cells. Embryonic stem cell clones were then microinjected into mouse blastocysts. Chimeric males, screened for their ability to transmit YACs via their gonads, were then bred with mice deficient in the production of murine antibodies. Culturing the two transgenic lines, one containing the human heavy chain locus and the other containing the human light chain locus, produces progeny that produce human antibodies in response to immunization.

General methods for cloning murine immunoglobulin variable regions are described, for example, in the disclosure of Orlandi et al Proc.Nat' l Acad.Sci USA 86-3833(1989), which is incorporated by reference in its entirety. Methods for making humanized MAbs are described in, e.g., Carter et al, proc.nat' l acad. sci USA 89: 4285(1992), and Singer et al J.Immun.150: 2844(1992), Mountain et al Biotechnol. Genet. Eng. Rev.10: 1(1992), and Coligan 10.19.1-10.19.11, each of which is hereby incorporated by reference.

Typically, the V κ (variable light chain) and V of the RS7 antibody_H(variable heavy chain) sequences can be obtained by a variety of molecular cloning methods, such as RT-PCR, 5' -RACE and cDNA library screening. Specifically, the VH and vk genes of MAb RS7 were cloned by RT-PCR and 5' -RACE, respectively, by PCR amplification from hybridoma cells and their sequences determined by DNA sequencing. To confirm its authenticity, cloned V was used_LAnd V_HThe gene is expressed in cell culture as a chimeric Ab, as described by Orlandi et al, (Proc. nat' l Acad. Sci USA, 86: 3833(1989)), which is incorporated by reference. Based on the V gene sequence, humanized RS7 antibodies were then designed and constructed as described by Leung et al (mol. Immunol., 32: 1413(1995)), which is incorporated by reference. cDNA can be prepared from any hybridoma or transfected cell line known to produce murine or chimeric RS7 antibodies by general Molecular Cloning techniques (Sambrook et al, Molecular Cloning, Alabortory manual, 2DED (1989)). In a preferred embodiment, the RS7 hybridoma line is used. The V.kappa.sequences for mAbs can be amplified using primers VK1BACK and VKIFOR (Orlandi et al, 1989) or extended primer sets as described by Leung et al (BioTechniques, 15: 286(1993), incorporated by reference), while V.kappa._HThe sequences may be amplified using the primer pair VH1BACK/VH1FOR (Orlandi et al, 1989 above), or primers annealing to the murine IgG constant region as described by Leung et al (Hybridoma, 13: 469(1994), incorporated by reference). The cDNA containing 10. mu.l of the first strand cDNA product, 10. mu.l of 10 XPCR buffer [500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl₂And 0.01% (W/V) gelatin](Perkin Elmer Cetus, Norwalk, CT), 250. mu.M of each DNTP, 200nM primer, and 5 units of Taq DNA polymerase (Perkin Elmer Cetus) were subjected to 30 PCR cycles. Each PCR cycle preferably includes denaturation at 94 ℃ for 1 minute, annealing at 50 ℃ for 1.5 minutes, and polymerization at 72 ℃ for 1.5 minutes. The amplified V.kappa.and VH fragments can be purified on 2% agarose (BioRad, Richmond, CA). Similarly, humanized V genes can be constructed by a combination of long oligonucleotide template synthesis and PCR amplification (as described by Leung et al (mol. Immunol, 32: 1413 (1995)).

The vk PCR product can be subcloned into a stating vector, such as pBR 327-based stating vector vk pBR, which contains an Ig promoter, a signal peptide sequence, and restriction sites to facilitate convenient in-frame ligation of the vk PCR product. The PCR products of the VH can be subcloned into a similar stabilizing vector, such as pBluescript-based VHpBS. Each clone containing each PCR product was available, and was incorporated by reference into Sanger et al proc.natl.acad.sci.usa, 74: 5463 (1977).

The DNA sequences described herein are to be understood as including all alleles, mutants and variants thereof, whether occurring naturally or induced.

The expression cassettes containing V.kappa.and VH, and promoter and signal peptide sequences were cleaved from V.kappa.pBR and VHpBS, respectively, and double-restriction cleaved into HindIII-BamHI fragments. The V.kappa.and VH expression cassettes are then ligated into suitable expression vectors, such as pKh and pGlg, respectively (Leung et al, Hybridoma, 13: 469 (1994)). Expression vectors can be co-transfected into appropriate cells, for example myeloma Sp2/0-Agl 4(ATCC, VA), selection for hygromycin resistant colonies, and preparation of chimeric or humanized RS7MAb in the supernatant assayed using, for example, an ELISA assay, as described below. Alternatively, the V.kappa.and VH expression cassettes may be assembled into modified staging vectors, V.kappa.pBR 2 and VHpBS2, cleaved into XbaI/BamHI and XhoI/BamHI fragments, respectively, and then subcloned into a single expression vector, pdHL2, Gilles et al (JImmunol. methods 125: 191 (1989)) and also described in Losman et al, cancer, 80: 2660(1997)) for expression in SP2/0-AGL4 cells. Another vector suitable for use in the present invention is the GS vector, such as Barnes et al, Cytotechnology 32: 109-123(2000), the vector is preferably expressed in NSO cell lines and CHO cells. Other suitable mammalian expression systems are described in Werner et al, Arzneim. -Forsch./Drug Res.48(11), Nr.8, 870-880 (1998).

Co-transfection was then performed as follows and clones secreting antibody were tested by ELISA. According to Co et al J immunol, 148: 1149(1992) approximately 10. mu.g of VKPkh (light chain expression vector) and 20. mu.g of VHpGIG (heavy chain expression vector) were used to transfect 5X 10 by electroporation⁶SP2/0 myeloma cells (BioRad, Richmond, CA). After transfection, 5% CO at 37 ℃ in complete HSFM medium (Life technologies, Inc., Grand Island, NY) can be performed on 96-well microtiter plates₂The cells are cultured. The selection step can be started 2 days later with the addition of hygromycin selection medium (Calbiochem, San Diego, Calif.) at a final concentration of 500 units/ml hygromycin. Cloning usually occurs 2-3 weeks after electroporation. The culture was then expanded for further analysis.

Suitable host cells include microbial or mammalian host cells. C6, which was constructed for the preparation of mabs, and other fusion proteins. Thus, a preferred embodiment of the present invention is a host cell comprising a DNA sequence encoding an anti-EGP-1 MAb, conjugate, fusion protein or fragment thereof. C6 cells (WO97/00326) were generated by transfecting primary human embryonic retinal cells with a plasmid containing the Adserotype 5(Ad 5) E1A and E1B coding sequences (Ad5 nucleotides459-3510) under the control of a human phosphoglycerate kinase (PGK) promoter. E1A and E1B are adenovirus early gene activation proteins 1A and 1B, respectively. The methods and compositions are particularly useful for generating stable expression of a desired human recombinant protein that is pre-translationally modified, for example, by glycosylation. C6 is particularly suitable as a host for recombinant protein production, e.g. c6 is a fully characterized human cell line and it has been developed for very good laboratory procedures. C6 can also be grown in suspension culture in serum-free medium devoid of any protein of human or animal origin and suitable for roller bottles, shake flasks, spinner flasks and bioreactors with a doubling time of about 35 hours. Finally, the presence of E1a resulted in up-regulation of gene expression under the control of the CMV enhancer/promoter while the presence of E13 prevented p 53-dependent apoptosis that could be enhanced by overexpression of the recombinant transgene. In one embodiment, the cell is capable of producing 2-200 times more recombinant protein and/or proteinaceous material than a conventional mammalian cell line.

Positive transfectoma clones secreting chimeric or humanized heavy chains were identified by ELISA assay. Briefly, a sample of supernatant from a hybridoma culture (100. mu.l) was added in triplicate with goat anti-human (GAH) -IgG, F (ab')₂Fragment-specific antibodies (JacksonImmunoresearch, West Grove, PA) were pre-coated on ELISA microtiter plates. The plates were incubated for 1 hour at room temperature. The plate was washed three times with washing buffer (PBS containing 0.05% polysorbate-20) to remove unbound protein. Horseradish peroxidase (HRP) -conjugated GAH-IgG, Fc fragment-specific antibody (Jackson ImmunoResearch, WestGrove, Pa.) was added to the wells, (100. mu.l of antibody diluted stock × 10⁴Supplemented with unbound antibody to a final concentration of 1.0 g/ml). After 1 hour incubation, the plates are typically washed three times. Reaction solution [ 100. mu.l of PBS containing 167. mu.g of o-phenylenediamine (OPD) (Sigma, St. Louis, Mo.), 0.025% hydrogen peroxide ] was added to each well]. Color development was carried out in the dark for 30 minutes. The reaction was stopped by adding 50. mu.l of 4M HCl solution to each well, and the absorbance at 490nm was measured using an automated ELISA reader (Bio-Tekinstruments, Winooski, VT). Bound chimeric antibodies were then determined relative to an unrelated chimeric antibody standard (available from Scotgen, ltd., Edinburg, Scotland).

The antibodies can be isolated from the cell culture medium as follows. Transfectoma cultures are adapted to serum-free media. To prepare chimeric antibodies, cells were grown to 500ml culture in roller bottles using HSFM. The culture was centrifuged to pellet the cells and the supernatant was filtered through a 0.2 μm membrane. The filtered medium was passed through a protein A column (1X 3cm) at a rate of 1 ml/min. The column was then washed with about 10 column volumes of PBS and protein a bound antibody eluted from the column with a buffer of 0.1M glycine (pH 3.5) containing 10mM EDTA. In the presence of 10. mu.l of 3M Tris (pH 8.6), 1ml of the eluted fraction was collected, and the protein concentration was determined by measuring the absorbance at 280/260 nm. The peak eluted fractions were pooled, dialyzed against PBS, and the protein was concentrated with Centricon 30(Amicon, Beverly, Mass.). Antibody concentrations were determined by ELISA and adjusted to about 1mg/ml with PBS as before. It is beneficial to add 0.01% (w/v) sodium azide as a preservative to the samples.

The nucleotide sequences of the primers used to prepare the RS7 antibody are listed in example 2 below. In a preferred embodiment, the humanized RS7 antibody or antibody fragment comprises the Complementarity Determining Regions (CDRS) of the murine RS7Mab and the Framework Regions (FRs) of the light and heavy chain variable regions of a human antibody and the light and heavy chain constant regions of a human antibody, wherein the CDRS of the light chain variable region of humanized RS7 comprise CDR1 comprising the amino acid sequence KASQDVSIAVA; a CDR2 comprising the amino acid sequence SASYRYT; and a CDR3 comprising the amino acid sequence QQHYITPLT; and the CDR of the heavy chain variable region of the humanized RS7MAb comprises CDR1 comprising the amino acid sequence NYGMN; a CDR2 comprising amino acid sequence WINTYTGEPTYTDDFKG and a CDR3 comprising amino acid sequence GGFGSSYWYFDV. Also preferably, the FRs of the light and heavy chain variable regions of the humanized antibody comprise at least one amino acid substituted by the corresponding FRs of murine RS7 Mab.

MAbs can be isolated and purified from hybridoma cultures using a variety of well-established methods. The separation methods include affinity chromatography using protein a sepharose, size exclusion chromatography and ion exchange chromatography. See, for example, Coligan, pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. See also, metals IN MOLECULAR BIOLOGY, vol.10, Baines et al, "Purification of Immunoglobulin G (IgG)" at pages 79-104 (The Humana Press, inc.1992).

The RS7MAb can be characterized using a variety of methods well known to those skilled in the art. For example, the ability of RS7 mabs to bind to RS7 antigen can be identified using indirect immunofluorescence detection, flow cytometric analysis, or Western analysis.

Preparation of RS7 antibody fragment

The invention relates to the use of RS7 and hRS7 antibody fragments. Antibody fragments recognizing specific epitopes can be prepared by known methodsAnd (4) preparing. Antibody fragments are antigen-binding portions of antibodies, e.g., F (ab')₂Fab', Fab, Fv, sFv, etc. Other antibody fragments include, but are not limited to: f (ab) 'produced by pepsin digestion of antibody molecules'₂And may be F (ab)'₂The disulfide bond of the fragment is reduced to prepare Fab' fragment. Such methods are described, for example, in golden berg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained herein, which are incorporated by reference in their entirety. Also, see Nisonoff et al, Arch biochem. Biophys.89: 230 (1960); porter, biochem.j 73: 119(1959), Edelman et al, METHODS IN EnzyrolyVol.1, pp.422 (Academic Press1967), and Coligan2.8.1-2.8.10 and 2.10-2.10.4. Alternatively, Fab' expression libraries can be constructed (Huse et al, 1989, Science, 246: 1274-. The invention includes antibodies and antibody fragments.

Single chain Fv molecules (scFVs) comprise a VL region and a VH region. The VL and VH regions combine to form a target binding site. The two regions may also be covalently linked by a peptide linker (L). An scFV molecule can be represented as either a VL-L-VH if the VL region is the N-terminal part of an scFV molecule or a VH-L-VL if the VH region is the N-terminal part of an scFV molecule. Methods for preparing scFV molecules and designing suitable peptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, r.rag and m.whitelow, "Single Chain fvs. 73-80(1995) and R.E.bird and B.W.Walker, "Single ChainAntibody Variable Regions," TIBTECH, Vol 9: 132, 137 (1991). These references are incorporated herein by reference in their entirety.

Antibody fragments can be prepared by proteolysis of full-length antibodies or expression of DNA encoding the fragment in e.coli (e.coli) or another host. Antibody fragments can be obtained by digesting the full length antibody with pepsin or papain using conventional methods. For example, an antibody fragment can be provided by enzymatic cleavage of the antibody with pepsin to provide a peptide represented as F (ab')₂The 5S fragment of (1). The fragment may also be treated with a thiol reducing agent, and optionally with a thiol reducing agentThe blocking agent for the sulfhydryl groups resulting from cleavage of the disulfide bonds is further cleaved to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using papain can directly produce two monovalent Fab fragments and an Fc fragment. Such methods are described, for example, in golden berg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references included herein, which are incorporated by reference in their entirety. Also, see Nisonoff et al, Arch biochem. biophysis.89: 230 (1960); porter, biochem.j.73: 119(1959), EDELMAN et al, METHODS INENZYMOLOGY Vol.1, pp.422 (Academic Press1967), and Coligan2.8.1-2.8.10 and 2.10-2.10.4.

Another form of antibody fragment is a peptide encoding a single Complementarity Determining Region (CDR). CDRs are fragments of the variable region of an antibody that are structurally complementary to, and more variable than, other portions of the variable region to which the antibody binds. Thus, CDRs are sometimes referred to as hypervariable regions. The variable region includes three CDRs. CDR peptides can be obtained by constructing genes encoding the CDRs of a desired antibody. The gene can be prepared, for example, by synthesizing the variable region from RNA of an antibody-producing cell using the polymerase chain reaction. See, for example, Larrick et al, Methods: a company to method Enzymology 2: 106 (1991); Courtenay-Luck, "genetic management of Mnonococcal antibodies", Monocolonnalantibiodies: RODUTION ENGINEERING AND CLINICAL APPLICATION, Ritter et al (eds.), page 166-179 (Cambridge University Press 1995); and Ward et al, "Genetic management and Expression of ANTIBODIES," simple ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS, Birch et al, (eds.), p 137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of the heavy chain to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen recognized by the intact antibody.

Preparation of chimeric, humanized and human RS7 antibody fusion proteins

Antibody fusion proteins and fragments thereof can be prepared by a variety of conventional methods including glutaraldehyde linkage between two functional groups to more specific linkages. The antibodies and/or antibody fragments are preferably covalently bound to each other, either directly or via a linking moiety, via one or more functional groups on the antibody or fragment, such as amine, carboxyl, phenyl, thiol, or hydroxyl groups. A variety of conventional linking agents other than glutaraldehyde may be used, such as diisocyanates, diisothiocyanates, bis (hydroxysuccinimide) esters, carbodiimides, maleimidohydroxysuccinimide esters, and the like.

A simple method of making chimeric, humanized and human RS7 antibody fusion proteins is to mix antibodies or fragments in the presence of glutaraldehyde to form an antibody fusion protein. The initial schiff base linking group may be stabilized by, for example, borohydride reduction to a secondary amine. Diisocyanates or carbodiimides may be used instead of glutaraldehyde as non-site specific linkers. The antibody fusion protein is expected to contain a higher binding specificity than the Mab because the fusion protein includes a portion that binds at least two epitopes of the RS7 antigen. Thus, antibody fusion proteins are a preferred form of RS7 antigen binding protein for use in therapy.

Herein, an antibody fusion protein comprises at least two chimeric, humanized or human RS7 mabs, or fragments thereof, wherein at least two mabs or fragments bind to different epitopes of RS7 antigen or an anti-RS 7 epitope and an epitope of a completely different antigen. For example, a bispecific RS7 antibody fusion protein can include a CEA antibody or fragment thereof and an RS7MAb or fragment thereof. The bispecific RS7 antibody fusion protein can be obtained, for example, by obtaining F (ab')₂Fragments were prepared as described above. Antibody F (ab')₂The interchain disulfide bonds of the fragments are slightly reduced by cysteine, with care taken to avoid light-heavy chain ligation, thereby forming Fab' -SH fragments. The thiol group is activated by an excess of bismaleimide linker (1, 1' - (methylenebis-4, 1-phenylene) bismaleimide). The RS7Mab was converted to Fab '-SH and then reacted with activated CEA Fab' -SH fragments to obtain bispecific RS7 antibody fusion proteins.

A multispecific RS7 antibody fusion protein can be obtained by adding an RS7 antigen-binding moiety to a bispecific chimeric, humanized or human RS7 antibody fusion protein. For example, a bispecific antibody fusion protein can be reacted with 2-iminothiolane to introduce one or more sulfhydryl groups for coupling the bispecific fusion protein to a third RS7 antigen MAb or fragment, using the bismaleimide activation method described above. These techniques for preparing antibody complexes are well known to those skilled in the art. See, for example, U.S. patent No. 4,925,648, which is incorporated by reference in its entirety.

Bispecific antibodies can be prepared by a variety of conventional methods, such as disulfide cleavage and reconstitution of intact IgG mixtures or, preferably, F (ab')₂Fragments, fusion of more than one hybridoma to form a polyoma capable of producing antibodies with more than one specificity, and by genetic engineering. Bispecific antibody fusion proteins have been prepared by oxidative cleavage of Fab' fragments from reductive cleavage of different antibodies. It is conveniently carried out by mixing two different F (ab') antibodies prepared by pepsin digestion of two different antibodies₂Fragments, reductive cleavage to form a mixture of Fab ' fragments, followed by oxidative reconstitution of disulfide bonds to produce F (ab ') comprising bispecific antibody fusion proteins containing Fab ' portions specific for each original epitope₂A mixture of fragments. General methods for preparing antibody fusion proteins can be found, for example, in Nisonoff et al, Arch biochem. Biophys.93: 470(1961), Hammering et al, j.exp.med.128: 1461(1968), and U.S. Pat. No. 4,331,647. The present invention relates to antibody fusion proteins or fragments thereof comprising at least one first anti-EGP-1 MAb or fragment thereof and at least one second MAb or fragment thereof other than the anti-EGP-1 MAb or fragment thereof of the present invention.

Highly selective attachment can be achieved by using heterobifunctional linkers such as maleimide hydroxysuccinimide esters. Reaction of the ester with the antibody or fragment will derivatize the amine group in the antibody or fragment, and then react this derivative with, for example, an antibody Fab fragment having a free thiol group (or, using, for example, a larger fragment to which a thiol group has been added or a Traut's reagent for the intact antibody). The linker is less likely to cross-link groups within the same antibody and provides selectivity of attachment.

It may be advantageous to attach the antibody or fragment at a site remote from the antigen binding site. This can be effected, for example, by linking to cleaved intrachain thiol groups, as described above. Another method includes reacting an antibody having an oxidized sugar moiety with another antibody having at least one free amine functional group. This forms a schiff base (imine) linkage, which is preferably stabilized by reduction to a secondary amine, for example by borohydride reduction, to form the final conjugate. For small molecules, the site-specific linkage is described in U.S. Pat. No. 4,671,958, while for large additions see U.S. Pat. No. 4,699,784-incorporated by reference.

ScFvs having linkers of more than 12 amino acid residues in length (e.g., 15-or 18-residue linkers) are capable of interacting with the VH and VL domains on the chain to form a mixture of monomers, dimers (known as diabodies) and small amounts of high molecular weight polymers in general (Kortt et al, Eur. J. biochem. (1994) 221: 151-157). However, ScFv with linkers more than 5 or less than 5 amino acid residues in length prevent intramolecular pairing of VH and VL regions on the same chain, forcing pairing of VH and VL regions on different chains. Linkers of 3-12 residues form mainly dimers (Atwell et al, Protein Engineering (1999) 12: 597-604). With a linker of 0-2 residues, trimers (called triabodies), tetramers (called tetrabodies) or higher oligomers of scFv will be formed; however, the precise mode of oligomerization appears to be dependent on composition and orientation of the V-zone in addition to linker length. For example, with a 0-residue linker, the scFv of anti-neuraminidase antibody NC10 forms predominantly trimers (VH to VL orientation) or tetramers (VL to VH orientation) (Dolezal et al, Protein Engineering (2000) 13: 565-. For construction of scFVs from NC10 with 1-and 2-residue linkers, the VH to VL orientation forms predominantly dimers (Atwell et al, Protein Engineering (1999) 12: 597-604); in contrast, the VL to VH direction forms a mixture of tetramers, trimers, dimers and higher molecular weight multimers (Dolezal et al, Protein Engineering (2000) 13: 565-. For the construction of scFVs in the VH-VL orientation of anti-CD 19 antibody HD37, the 0-residue linker forms only a trimer and the 1-residue linker forms only a tetramer (Le Gall et al, FEBS Letters (1999) 453: 164-.

The RS7 antibodies and fragments thereof of the invention can also be used to make antigen-specific diabodies, triabodies and tetrabodies, which are multivalent but monospecific. Non-covalent association of two or more scFv molecules can form functional diabodies, triabodies, and tetrabodies. A homodimer of monospecific diabodies identical scfvs, wherein each scFv comprises a VH region from a selected antibody that is linked to the same antibody by a short linker. The dimer is a bivalent dimer formed by non-covalent association of two scFV, resulting in two Fv binding sites. Trisomy results from the formation of a trivalent trimer of three scFvs, yielding three binding sites, while tetrasome results from a tetravalent tetramer of four scFvs, yielding four binding sites. Certain monospecific diabodies are prepared by using expression vectors comprising a recombinant gene construct comprising V_H1linker-V_L1See Holliger et al, proc.natl.acad.sci.usa 90: 6444 6448 (1993); a twell et al, molecular immunology 33: 1301, 1302 (1996); holliger et al, Nature Biotechnology 15: 632-631 (1997); helfrich et al, int.J. cancer 76: 232-239 (1998); kipriyanov et al, int.J. cancer 77: 763 772 (1998); holiger et al, Cancer Research 59: 2909-2916(1999)). Methods for constructing scFVs are disclosed in US-4,946,778(1990) and US-5,132,405 (1992). Methods for the preparation of multivalent, monospecific binding proteins based on scFVs are disclosed in US-5,837,242(1998), US-5,844,094(1998) and WO-98/44001 (1998). A preferred embodiment of the invention is a multivalent, multispecific antibody or fragment thereof comprising one or more antigen binding sites having affinity for an EGP-1 target antigen and one or more hapten binding sites having affinity for a hapten molecule.

Determination of antibody binding affinity

Relative junctions of the thus isolated mRS7, cRS7 and hRS7 antibodiesAvidity can be determined by direct radioimmunoassay. RS7 available¹³¹I or¹²⁵I is labeled by the chloramine-T method (see, e.g., Greenwood et al, biochem. J., 89: 123(1963), which is incorporated by reference). The specific activity of the iodinated antibody is typically adjusted to about 10. mu. Ci/. mu.g. Unlabeled and labeled antibodies were diluted to appropriate concentrations with reaction medium (HSFM supplemented with 1% horse serum and 100. mu.g/ml gentamicin). The appropriate concentrations of labeled and unlabeled antibodies were added together to the reaction tube in a total volume of 100. mu.l. ME180 cells (human cervical cancer cell line) were sampled and cell concentration was determined. The culture was centrifuged and the once washed cells were collected in the reaction medium and then vortexed in the reaction medium to a final concentration of about 10⁷Cells/ml. All steps were carried out at 4 ℃ under cold conditions. 100 μ l of the cell suspension was added to the reaction tube. The reaction was carried out at 4 ℃ for 2 hours under cold conditions, during which the reaction tube was gently shaken periodically to resuspend the cells. After the reaction period, 5ml of washing buffer (PBS containing 1% BSA) was added to each tube. The suspension was centrifuged and the cell pellet was washed a second time with 5ml of wash buffer. After centrifugation, the amount of residual radioactivity remaining in the cell pellet was counted in a gamma counter (Minaxi, PackardInstruments, Sterling, Va.).

Expression vector

An expression vector is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of specific regulatory elements including constitutive or inducible promoters, tissue-specific regulatory elements and enhancers. The gene is said to be "operably linked" to a regulatory element. A promoter is a DNA sequence that directs transcription of a resulting gene. Structural genes are DNA sequences that are transcribed into messenger rna (mrna), which is then translated into the amino acid sequence characteristic of a particular polypeptide. Typically, a promoter is located in the 5' region of a gene, near the transcription start site of a structural gene. If the promoter is an inducible promoter, the rate of transcription increases in response to an inducing agent. Conversely, if the promoter is a constitutive promoter, the rate of transcription is not regulated by an inducing agent. An enhancer is a DNA regulatory element that increases the efficiency of transcription regardless of the distance or orientation of the enhancer relative to the transcription start site.

An isolated DNA molecule is a DNA fragment that is not integrated into the genomic DNA of an organism. For example, the cloned RS7 antigen gene is a DNA fragment isolated from genomic DNA of mammalian cells. Another example of an isolated DNA molecule is a chemically synthesized DNA molecule that is not integrated into the genomic DNA of an organism. Complementary DNA (cdna) is a single-stranded DNA molecule formed from an mRNA template by the enzyme reverse transcriptase. Typically, primers complementary to portions of the mRNA are used to initiate reverse transcription. The skilled artisan also uses the term "cDNA" to refer to a double-stranded DNA molecule consisting of the single-stranded DNA molecule and its complementary DNA strand.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, or phage, that has the ability to replicate autonomously in the host cell. Cloning vectors typically contain one or a small number of restriction enzyme recognition sites at which exogenous DNA sequences can be inserted in a determinable fashion without loss of the essential biological function of the vector, and a marker gene suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance. The recombinant host may be any prokaryotic or eukaryotic cell containing a cloning vector or an expression vector. The term is also meant to include prokaryotic or eukaryotic cells that have been genetically engineered to contain a cloned gene in the chromosome or genome of the host cell. The term expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mNA and translation of the mRNA into one or more polypeptides.

Use of humanized, human and chimeric RS7 antibodies in therapy and diagnosis

The present invention relates to a method of diagnosing or treating a malignancy in an individual comprising administering to the individual a therapeutically effective amount of a therapeutic conjugate comprising an EGP-1MAb or fragment thereof or an antibody fusion protein or fragment thereof, wherein the EGP-1MAb or fragment thereof or antibody fusion protein or fragment thereof is conjugated to at least one therapeutic agent and subsequently formulated in a pharmaceutically acceptable excipient. It is also contemplated that unconjugated (naked) EGP-1MAb or fusion constructs with other antigen-binding moieties may also be used to treat EGP-1 expressing cancer cells. These unbound antibodies can be conveniently combined with other therapeutic modalities, such as chemotherapy, radiation therapy and/or immunotherapy, either together or in various sequences and procedures. Also preferred is a method of diagnosing or treating cancer comprising: administering to an individual in need thereof a multivalent, multispecific antibody or fragment thereof comprising one or more antigen binding sites for EGP-1 antigen and one or more hapten binding sites, waiting a sufficient amount of time for the amount of non-binding protein to clear from the blood stream of the individual; the individual is then administered a carrier molecule comprising a diagnostic agent, a therapeutic agent, or a combination thereof, which binds to the binding site of the antibody. In a preferred embodiment, the cancer is lung, breast, head and neck, ovarian, prostate, bladder or colon cancer.

The hybridoma technology for producing monoclonal antibodies (MAbs) has passed through a method for producing molecular probes that localize or kill cancer cells. Tumor imaging techniques using radiolabeled mabs have been used to describe cancer infiltration in a variety of malignancies. In experimental animals and humans, antibodies and other targeting antibodies have been used for radioimmunoassay of carcinoembryonic antigen in a variety of tumors expressing carcinoembryonic, as well as tumors such as melanoma, colon and breast cancers. Golden berg et al, Cancer res.40: 2984 (1980); hwang et al, cancer Res.45: 4150 (1985); zalcberg et al, J.nat' l.cancer Inst.71: 801 (1983); colcher et al, Cancer Res.43: 736 (1983); (Larson et al, J.Nucl. Med.24: 123 (1983); Delind et al, Cancer Res.40: 3046 (1980); Epenetos et al, Lancet 2: 999 (1982)).

The use of MAbs in vitro diagnostics is well known. See, e.g., Carlsson et al, Bio/Technology 7 (6): 567(1989). For example, MAbs can be used to detect the presence of tumor associated antigens in tissue of a biological sample. MAbs can also be used to detect the amount of tumor associated antigen in clinical fluid samples using techniques such as radioimmunoassay, enzyme-linked immunosorbent assay and fluoroimmunoassay.

Conjugates of tumor-targeting MAbs and toxins are useful for selectively killing cancer cells in vivo (Spalding, Bio/Technology 9 (8): 701 (1991); golden amber Science & Medicine 1 (1): 64 (1994)). Therapeutic studies in experimental animal models, for example, have demonstrated the anti-tumor activity of antibodies carrying cytotoxic radionuclides. (Goldenberg et al, Cancer Res.41: 4354(1981), Cheung et al, J.Nat' l Cancer Inst.77: 739(1986), and Senekowitsch et al, J.Nucl. Med.30: 531 (1989)). See also Stein et al, Antibody Immunocon j. radiopharm.4: 703(1991), which is incorporated by reference in its entirety. Moreover, phase I therapeutic trials using certain of the mabs have begun for the treatment of lymphoma, melanoma, and other malignancies. See, e.g., denadro et al, int.j. cancer supply.3: 96(1988), and golden berg et al, j.clin.oncol.9: 548(1991).

Humanized, chimeric and fully human antibodies and fragments thereof are useful in diagnostic and therapeutic methods. Accordingly, the invention includes a method of delivering a diagnostic or therapeutic agent, or a combination thereof, to a target comprising (i) providing a composition comprising an anti-EGP-1 antibody and (ii) administering a diagnostic or therapeutic antibody conjugate to an individual in need thereof. Preferably, the chimeric, humanized and fully human RS7 antibodies and fragments thereof of the invention are used in methods of treating malignancies.

Also described herein are cancer cell-targeting diagnostic or therapeutic conjugates comprising an antibody component comprising an anti-EGP-1 MAb or fragment thereof or an antibody fusion protein or fragment thereof that binds to cancer cells, wherein the antibody component is bound to at least one diagnostic agent or at least one therapeutic agent. Preferably, the diagnostic conjugate comprises at least a photoactivatable diagnostic agent or an MRI contrast agent. More preferably, the diagnostic agent is a radioactive label with an energy of 60-4,000 keV.

The composition for use in therapy comprises at least one naked or conjugated humanized, chimeric or human RS7 antibody alone or in combination with other naked or conjugated humanized, chimeric, human or other antibodies of the invention or in combination with other naked or conjugated humanized, chimeric or human antibodies not disclosed herein. The invention also encompasses the administration of the bound or naked antibody with a therapeutic agent that does not bind to the anti-EGP-1 antibody, such as an immunomodulator, or a diagnostic agent. Naked or conjugated antibodies directed against the same or different epitopes or antigens may also be combined with one or more antibodies of the invention.

Thus, the present invention relates to the administration of anti-EGP-1 antibodies and fragments thereof alone, as naked antibodies or antibody fragments, or as a multi-therapy. Preferably, the antibody is a humanized, chimeric or fully human RS7 antibody or fragment thereof. Part of the multiplex therapy also includes immunotherapy with naked anti-EGP-1 antibodies, with the addition of other antibodies administered in the form of naked antibodies, fusion proteins, or immunoconjugates. For example, a humanized, chimeric or fully human RS7 antibody can be combined with another naked humanized, chimeric RS7 or other antibody, or a humanized, chimeric RS7 or other epitope bound to an isotope, one or more chemotherapeutic agents, cytokines, toxins, or combinations thereof. For example, the invention contemplates treatment of naked or conjugated EGP-1 or RS7 antibodies or fragments thereof prior to, in combination with, or following administration of other solid tumor/cancer associated antibodies, such as anti-EGP-2, CEA, CSAP, MUC1-4, EGFR, HER2/neu, PSA, CC49 (anti-Tag 72 antibody) and PSMA antibodies. These solid cancer antibodies may be naked or conjugated with, inter alia, drugs, enzymes, hormones, toxins, isotopes or immunomodulators. Fusion proteins of humanized, chimeric or fully human RS7 antibodies and toxins may also be used in the present invention. A variety of different antibody combinations can be constructed, either as naked antibodies or as partially naked and partially conjugated to a therapeutic agent or immunomodulator. Alternatively, different naked antibody combinations may be used for administration in combination with other therapeutic agents, such as cytotoxic drugs or with radiation. Combinations of the antibodies may also be prepared using, advantageously, antisense oligonucleotides as are known in the art. As such, the therapeutic conjugates also include oligonucleotides, particularly antisense oligonucleotides, preferably useful against cancer genes and oncogene products of B-cell malignancies. For example, antisense molecules that inhibit the expression of bc1-2, which are described in U.S. Pat. No. 3, 5,734,033(Reed), incorporated by reference in its entirety, are also conjugated to or form the therapeutic portion of an antibody fusion protein or administered with the humanized RS7 antibody of the present invention.

The direct targeting of monospecific binding proteins linked to diagnostic or therapeutic agents described herein to RS7 positive tumors. Monospecific molecules bind selectively to the targeted antigen and as the number of binding sites on the molecule increases, the affinity for the targeted cell increases and a longer residence time is observed at the desired location. Furthermore, non-antigen binding molecules can be cleared rapidly from the body while minimizing normal tissue exposure. The use of multispecific binding proteins pretarges RS7 positive tumors for subsequent specific delivery of diagnostic or therapeutic agents. The agent is carried by succinylglycerol Histamine (HSG) containing peptide. The murine monoclonal antibody named 679(IgG1, K) binds with high affinity to HSG containing the tripeptide moiety (Morel et al, Molecular immunology, 27, 995-1000, 1990). 679 the MAb can bind to hRS7 which can bind to HSG and target antigen to form a bispecific binding protein. Selective haptens can also be used. These binding proteins bind selectively to the targeted antigen to produce increased affinity and longer residence time at the desired location. Furthermore, non-antigen binding antibodies can be cleared rapidly from the body while minimizing normal tissue exposure.

The RS7 antibodies and fragments thereof are useful for treating mammalian disorders such as cancer. Cancers include, but are not limited to, lung, breast, bladder, ovarian, prostate, and colon cancers.

Delivering a diagnostic or therapeutic agent to a target site for diagnosis or treatment according to the present invention includes providing an anti-EGP-1 antibody or fragment thereof containing the diagnostic or therapeutic agent and administering the binding protein to an individual in need thereof. Diagnosis also requires the step of detecting the binding protein using known techniques.

The antibodies and fragments thereof of the invention and the diagnostic or therapeutic agent can be administered intravenously, intra-arterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, intrathecally, or regionallyInfusion of a tube or direct intra-traumatic injection is administered to the mammal. When the binding protein is administered by injection, it may be administered as a continuous infusion or as a single or multiple bolus injection. 20-800 mg/m for treatment²The dosage of (A) is proper, preferably 100-500 mg/m²While the same low dose is recommended for diagnostic imaging, e.g., 0.5mg to 100mg per patient. The dose may be repeated at different frequencies depending on the clinical situation and the tolerance level of the patient.

The antibody and diagnostic or therapeutic agent may be provided as a kit for therapeutic and diagnostic use in humans or mammals in a pharmaceutically acceptable carrier, preferably Phosphate Buffered Saline (PBS) at physiological pH and concentration. The formulation is preferably sterile, especially if it is intended for use in humans. Optional components of the kit include stabilizers, buffers, labeling agents, radioisotopes, paramagnetic compounds, secondary antibodies for enhanced clearance and conventional syringes, columns, vials, and the like.

Naked antibody therapy

A therapeutically effective amount of the naked chimeric, humanized and fully human RS7 antibody, or fragment thereof, can be formulated in a pharmaceutically acceptable excipient. The efficacy of naked chimeric, humanized and fully human RS7 antibodies can be enhanced by supplementing these naked antibodies with one or more other naked antibodies, immunoconjugates of one or more chimeric, humanized and fully human RS7 antibodies conjugated to a therapeutic agent such as a drug, toxin, immunomodulator, hormone, growth factor, enzyme, oligonucleotide or therapeutic radionuclide, or one or more therapeutic agents comprising a drug, toxin, immunomodulator, hormone, growth factor, enzyme, oligonucleotide or therapeutic radionuclide, administered simultaneously or sequentially with the RS7 antibody or fragment thereof or according to a prescribed dosage regimen.

In a preferred embodiment, the naked or conjugated RS7 antibody of the invention is combined with at least one cancer drug. Such combination therapy can enhance the efficacy of the drug or lower drug doses required. For example, determination of Dox-RS7 and 2P-Dox-RS7 against the Lung cancer cell line, C, respectivelyalu3, and IC of two breast cancer cell lines, MDA468 and T47D₅₀The value is obtained. Calu3 and T47D cells were positive for EGP-1 antigen and negative for CEA antigen, while MDA468 was positive for both EGP-1 and CEA antigens. The results show the IC of Dox-RS7₅₀IC of 2P-Dox-RS7 with a value of 0.04. mu.g/ml₅₀The value was 0.023. mu.g/ml. Thus, conjugation of the naked, human, humanized or chimeric anti-EGP-1 antibodies or fragments of the invention to a specific drug, such as 2P-Dox, can help overcome multi-drug resistance. However, it is also possible when the antibody is combined with a specific drug, as described above.

RS7 immunoconjugates

The invention also relates to the use of humanized, chimeric and human RS7 antibodies and fragments thereof for therapy. The goal of immunotherapy is to deliver cytotoxic doses of radiation, toxins, cytokines, enzymes, or hormones or drugs to target cells while minimizing exposure of non-target tissues. The RS7 antigen-binding protein can be used for treating various tumors, such as tumors of lung, breast, bladder, ovary, uterus, stomach and prostate.

Any of the antibodies or antibody fusion proteins of the invention and fragments thereof can be conjugated to one or more therapeutic or diagnostic agents. Typically, one therapeutic or diagnostic agent is bound to each antibody or antibody fragment and more than one therapeutic or diagnostic agent may be conjugated to the same antibody or antibody fragment. If the Fc region is not present (e.g., when the antibody used as the antibody component of the immunoconjugate is an antibody fragment), it is possible to introduce a sugar moiety into the light chain variable region of the full length antibody or antibody fragment. See, e.g., Leung et al, R Immunol.154: 5919 (1995); hansen et al, U.S. Pat. No. 5,443,953(1995), Leung et al, U.S. Pat. No. 6,254,868, all of which are incorporated herein by reference in their entirety.

The engineered saccharide moieties can be used to conjugate therapeutic or diagnostic agents. Methods for conjugating peptides to antibody components via antibody carbohydrate moieties are well known to those skilled in the art. See, e.g., Shih et al, int.J.cancer 41: 832 (1988); shih et al, int.J.cancer 46: 1101 (1990); and Shih et al, U.S. Pat. No. 5,057,313, all of which are incorporated herein by reference in their entirety. The general method involves reacting an antibody component containing oxidized sugar moieties with a carrier polymer containing at least one free amine functional group and then loading a plurality of peptides. This reaction forms a schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate. Also, the antibody can be conjugated to a chelator such as DTPA (e.g., Mx-DTPA), DOTA, TETA, or NOTA.

The antibody fusion protein of the present invention includes two or more antibodies or fragments thereof and each antibody or fragment constituting the fusion protein may comprise a therapeutic agent or a diagnostic agent. In addition, one or more antibodies or fragments of the antibody fusion protein may contain more than one conjugated therapeutic or diagnostic agent. Furthermore, the therapeutic agents need not be the same but may be different, for example, a drug and a radioisotope may be conjugated to the same fusion protein. In particular, IgG may be used¹³¹I radiolabeled and bound to a drug.¹³¹I can be incorporated into tyrosine of IgG and drugs attached to the epsilon amino group of lysine of IgG. Both the therapeutic and diagnostic agents may also be conjugated to reduced sulfhydryl groups and to sugar side chains.

A variety of diagnostic and therapeutic agents may be conveniently conjugated to the antibodies of the invention. The therapeutic agents cited herein are those agents that are also useful for administration separately from the naked antibody described above. Therapeutic agents include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotic agents, particularly doxorubicin, methotrexate, paclitaxel, CPT-11, camptothecin and other drugs from these and other classes of anticancer drugs and the like, methylhydrazine derivatives, adrenocortical suppressants, antagonists, endothelial somatostatin, paclitaxel and the like.

Other cancer chemotherapeutic drugs suitable for use in preparing immunoconjugates and antibody fusion proteins include nitrogen mustards, aziridine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, tyrosine kinase inhibitors such as those that inhibit EGF-receptor tyrosine kinase, BCR ABL tyrosine kinase or VEGF-body tyrosine kinase, and the like. Suitable chemotherapeutic agents are described in REMINGTON' S pharmaeutical SCIENCES, 19TH Ed. (Mack Publishing Co.1995) in GOODMAN and GILMAN' STHE PHHE PHARMACOLOGICAL BASIS OF THERAPEUTIC, 7th Ed. (MacMillan Publishing Co.1985) and revisions OF these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those skilled in the art.

Toxins such as pseudomonas exotoxin can also be complexed to or form the therapeutic agent portion of the immunoconjugates of the RS7 and hRS7 antibodies of the invention. Other toxins suitable for use in preparing the conjugates or other fusion proteins include ricin, abrin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-a, pokeweed antiviral protein, gelonin, diphtheria toxin, pseudomonas exotoxin, and pseudomonas endotoxin. See, e.g., Pastan et al CELL 47: 641(1986), and golden berg, CA-A Cancer Journarfor Clinicanins 44: 43(1994).

Other toxins suitable for use in the present invention are known to those skilled in the art and are described in U.S. Pat. No. 6,077,499, which is incorporated by reference in its entirety.

Immunomodulators, such as cytokines, can also be administered in conjunction with or form the therapeutic portion of EGP-1, RS7 and hRS7 immunoconjugates, or without binding to the chimeric, humanized or human RS7 antibodies or fragments thereof of the present invention. As used herein, the term "immunomodulator" includes cytokines, stem cell growth factors, lymphotoxins, such as Tumor Necrosis Factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferon- α, - β, and- γ), erythropoietin, thrombopoietin or combinations thereof. Examples of suitable immunomodulator moieties include IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, and combinations thereof, and interferon-gamma, TNF-alpha, and the like. Alternatively, the individual may receive a naked EGP-1 or RS7 antibody and a separately administered cytokine, which may be administered prior to, concurrently with, or subsequent to the administration of the naked RS7 antibody. As described above, the RS7 antibody can also bind to an immunomodulator. The immunomodulator may also bind to a hybrid antibody containing one or more antibodies that bind to different antigens.

The therapeutic or diagnostic agent may be linked at the hinge region of the reduced antibody component by the formation of disulfide bonds. Alternatively, the peptido-antibody may be linked using a heterobifunctional cross-linker, such as N-succinyl 3- (2-pyridyldithio) propionate (SPDP). Yu et al, int.j.cancer, 56: 244(1994). General methods for such bonding are well known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); upeslaciis et al, "Modificationof Antibodies by Chemical Methods," in MONOCLONALANTIBIDIES: PRINCIPLES AND application, Birch et al (eds.), page 187-230 (Wiley-Liss, Inc. 1995); price, "Production and propagation of Synthetic Peptide-Derived Antibodies, MonoC NAL Antibodies: PRODUCTION, ENGINER-ING AND CLINICALAPPLICATION, Ritter et al (eds.), pages 60-84 (Cambridge university Press 1995). Alternatively, the therapeutic or diagnostic agent may be bound via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group may be used to increase loading of the same peptide to which the thiol group is bound, or the carbohydrate group may be used to bind a different peptide.

In addition, the radiolabeled antibody, immunoconjugate, or fragment thereof may include a gamma-emitting isotope or positron emitter for diagnostic imaging. Suitable radioisotopes, especially those having an energy of 25 to 4,000keV, include¹³¹I，¹²³I，¹²⁴I，⁸⁶Y，⁶²Cu，⁶⁴Cu，⁶⁷Ga，⁶⁸Ga，^99mTc，^94mTc，¹⁸F，¹¹C，¹³N，¹⁵O，⁷⁶Br, and the like. See, for example, U.S. patent application entitled "Labeling targeting Agents with Gallium-68" to G.L.Griffiths and W.J.McBride (U.S. provisional application No. 60/342,104), which discloses positron emitters for imaging purposes, such as¹⁸F，⁶⁸Ga，^99mTc, etc., which application is incorporated by reference in its entirety. Preferably, the energy of the diagnostic and therapeutic radionuclide is 25-4,000 keV. Other suitable radionuclides include⁹⁰Y，¹¹¹In，¹²⁵I，³H，³⁵S，¹⁴C，¹⁸⁶Re，¹⁸⁸Re，¹⁸⁹Re，¹⁷⁷Lu，⁶⁷Cu，²¹²Bi，²¹³Bi，²¹¹At，¹⁹⁸Au，²²⁴Ac，¹²⁶I，¹³³I，⁷⁷Br，^113mIn，⁹⁵Ru，⁹⁷Ru，¹⁰³Ru，¹⁰⁵Ru，¹⁰⁷Hg，²⁰³Hg，^94mTc，^121mTe，^122mTe，^125mTe，¹⁶⁵Tm，¹⁶⁷Tm，¹⁶⁸Tm，¹¹¹Ag，¹⁹⁷Pt，¹⁰⁹Pd，³²P，³³P，⁴⁷Sc，¹⁵³Sm，¹⁷⁷Lu，¹⁰⁵Rh，¹⁴²Pr，¹⁴³Pr，¹⁶¹Tb，¹⁶⁶Ho，¹⁹⁹Au，⁵⁷CO，⁵⁸Co，⁵¹Cr，⁵⁹Fe，⁵⁸F，⁷⁵Se，²⁰¹Tl，²²⁵Ac，⁷⁶Br，⁸⁶Y，¹⁶⁹Yb，¹⁶⁶Dy，²¹²Pb and²²³Ra。

for example⁶⁷Cu, which is considered to be a better radioisotope for immunotherapy due to its 61.5 hour half-life and abundant supply of beta and gamma rays, can be bound to RS7 antigen binding protein with the chelator p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase, supra. Alternatively, 90Y emitting high energy beta particles can be conjugated to RS7 antigen binding protein with diethylenetriaminepentaacetic acid (DTPA).And, for direct use¹³¹I methods for radiolabeling RS7 mabs are described in Stein et al (1991), supra, and Govindan et al, WO 9911294A1 entitled "Stable Radiodine Conjugates and methods for therir Synthesis," which is incorporated herein by reference in its entirety.

The RS7 antibody or fragment thereof of the invention containing a boron addend-loaded carrier for thermal neutron activation therapy will function normally in a similar manner. However, it would be beneficial to wait until targeted immunoconjugate clearance occurs before neutron irradiation is performed. The use of antibodies that bind to RS7 antibody can accelerate clearance. See us 4,624,846 for a description of this general principle. For example, a boron addend such as carborane can be attached to the RS7 antibody. Carboranes can be prepared using carboxyl functionality on the side chain, as is well known in the art. Attachment of the carborane to a support, such as an aminodextran, can be achieved by activating the carboxyl group of the carborane and condensing with an amine on the support to produce an intermediate conjugate. The intermediate conjugate is then bound to the RS7 antibody. After administration of the RS7 antibody conjugate, the boron addend is activated by irradiation with challenge neutrons to convert the radioactive atom, which decays by alpha emission to produce a highly toxic, short-range effect.

In addition, the invention includes methods of diagnosing cancer in an individual. Diagnosis can be carried out by administering a diagnostically effective amount of a diagnostic conjugate formulated with a pharmaceutically acceptable excipient and then detecting the label. For example, radioactive and non-radioactive agents may be used as diagnostic agents. Suitable non-radioactive diagnostic agents are contrast agents suitable for magnetic resonance imaging, computed tomography or ultrasound. Magnetic imaging agents include, for example, nonradioactive metals such as manganese, iron, and gadolinium, which, when used with the antibodies of the present invention, are complexed with metal-chelator compositions including 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs. See U.S. serial No. 09/921,290 filed on 10/2001, which is incorporated by reference in its entirety.

Thus, a method of diagnosing a malignancy in an individual is described, comprising (i) performing an in vitro diagnosis on a sample from the individual with a composition comprising a naked anti-EGP-1 MAb or fragment thereof or a naked antibody fusion protein or fragment thereof. For example, RT-PCR and immunodetection in vitro diagnostic methods are useful as diagnostic/detection methods for detecting the transient amount of EGP-1 in tissues, blood and other body fluids. Immunohistochemistry can be used to detect the presence of EGP-1 in cells or tissues. Preferably, the malignancy being diagnosed is cancer. Most preferably, the cancer is selected from lung, prostate, ovarian, breast, colon and bladder cancer.

In addition, a detectable label, such as a fluorescent molecule, or a cytotoxic agent, such as a heavy metal or radionuclide, can be conjugated to a chelator such as DTPA, DOTA, TETA or NOTA or a suitable peptide. For example, therapeutically effective immunoconjugates can be obtained by binding a photoactivator or dye to an antibody fusion protein. Fluorescent compositions, such as fluorescent dyes, and other chromogens, or dyes, such as porphyrins, which are sensitive to visible light, have been used to detect and treat lesions by directing appropriate light at the lesion. In THERAPY, it is known as light radiation, phototherapy or PHOTODYNAMIC THERAPY (Jori et al (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, chem. Britain 22: 430 (1986)). In addition, monoclonal antibodies can also be conjugated to photoactivated dyes to effect light therapy. Mew, J.Immunol.130: 1473 (1983); idem, Cancer res.45: 4380 (1985); oseroff et al, Proc.Natl.Acad.Sci.USA 83: 8744 (1986); idem, photochem. photobiol.46: 83 (1987); hasan et al, prog.clin.biolres.288: 471 (1989); tatsuta et al, Lasers surg. med.9: 422 (1989); pelegrin et al, Cancer 67: 2529(1991). However, these early studies did not include the use of endoscopic therapy, particularly the use of antibody fragments or subfragments. Thus, the present invention relates to the therapeutic use of immunoconjugates comprising a photoactivator or a dye.

Contrast agents such as MRI contrast agents, paramagnetic beads and ultrasound enhancers are also contemplated by the present invention. For example, gadolinium ions, lanthanum ions, manganese ions or other comparable labels, CT contrast agents, and ultrasound contrast agents are suitable for use in the present invention. In a preferred embodiment, the ultrasound enhancing agent is a liposome comprising a humanized RS7 IgG or fragment thereof. Also preferably, the liposomes are gas filled.

For therapeutic purposes, the RS7 antibodies and fragments thereof of the invention are administered to a patient in a therapeutically effective amount. An antibody is said to be administered in a "therapeutically effective amount" if the amount administered has a physiologically significant meaning. An agent is physiologically significant if its presence results in a physiologically detectable change in the recipient mammal.

In vitro diagnosis

The invention relates to RS7 antibodies, including RS7 and hRS7 antibodies and fragments thereof, and application thereof in vitro detection of the presence of RS7 antigen in biological samples. In such immunoassays, the RS7 antibody can be used in the liquid phase or bound to a solid support, as described below. See also, Stein et al (1993), supra, and Stein et al, Cancer res.49: 32(1989), which is incorporated herein by reference in its entirety.

An example of a detection method for determining the release of an antigen comprising RS7 from a biological sample is a Radioimmunoassay (RIA). For example, in one form of RIA, the test agent is mixed with RS7 antigen in the presence of radiolabeled RS7 antigen. In this method, the test substance concentration will be inversely proportional to the amount of labeled RS7 antigen bound to the Mab and directly related to the amount of free labeled RS7 antigen. Other suitable detection methods will be apparent to those skilled in the art.

Alternatively, an in vitro assay comprising RS7 antigen binding protein bound to a solid support can be performed. For example, mabs may be attached to a polymer, such as aminodextran, so that the mabs are attached to an insoluble support, such as a polymer-coated bead, plate or tube.

Other suitable in vitro assays will be apparent to those skilled in the art. The specific concentrations of detectably labeled RS7 antigen binding protein and RS7 antigen, incubation temperature and time, and other assay conditions may vary depending on a number of factors including the concentration of RS7 antigen in the sample, the nature of the sample, and the like. The binding activity of the RS7 antigen binding protein sample can be determined according to well known methods. The skilled artisan, by employing routine experimentation, will be able to determine the operability and optimal conditions for each assay.

Other steps such as washing, stirring, shaking, filtering, etc. may be added to the assay, as is customary or desirable in a particular environment.

An enzyme-linked immunosorbent assay (ELISA) can be used to determine the presence of RS7 antigen in a biological sample. In a direct competitive ELISA, a pure or semi-pure antigen preparation is bound to a solid support that is insoluble in the liquid or cell extract to be detected and then an amount of detectably labeled soluble antibody is added to detect and/or quantify the binary complex formed between the solid phase antigen and the labeled antibody.

In contrast, a "dual determinant" ELISA, also known as a "two-site ELISA" or "sandwich assay", requires a small amount of antigen and the assay does not require extensive purification of the antigen. Therefore, a direct competitive ELISA for detection of antigens in clinical samples is preferred to a dual determinant ELISA. See, for example, "the use of the double-reagent ELISA for quantification of the C-MYC oncoprotein in biopsys specs", Field et al, Oncogene 4-1463 (1989); spandidos et al, AntiCancerRes.9. 821(1989).

In a dual determinant ELISA, an amount of unlabeled MAb or antibody fragment ("capture antibody") is bound to a solid support, the test sample is contacted with the capture antibody, and then an amount of detectably labeled soluble antibody (antibody fragment) is added to detect and/or quantify the ternary complex formed between the antibody, antigen, and labeled antibody. Antibody fragments are parts of antibodies such as F (ab')₂，F(ab)₂Fab', Fab and the like. Herein, an antibody fragment is a portion of RS7Mab that binds to an epitope of RS7 antigen. The term "antibody fragment" also includes any synthetic or genetically engineered complex formed by binding to a particular antigenProteins that function in a manner similar to antibodies. For example, antibody fragments include isolated fragments comprising a light chain variable region, an "Fv" fragment comprising the variable regions of a heavy or light chain, and a recombinant single chain polypeptide molecule, wherein the light and heavy chain variable regions are linked via a peptide linker. An antibody fusion protein is a multispecific antibody composition comprising at least two substantially monospecific antibodies or antibody fragments, wherein at least two antibodies or antibody fragments bind to different epitopes of the RS7 antigen. RS7 fusion proteins also include conjugates of the antibody fusion proteins with diagnostic or therapeutic agents. The term RS7 antibody includes humanized, chimeric, human and murine antibodies, antibody fragments thereof, immunoconjugates and fragments thereof and antibody fusion proteins and fragments thereof.

Methods for performing dual determinant ELISAs are well known. See, for example, Field et al, supra, Spandidos et al, supra, and Moore et al, "Twin-Site ELISAs for fos and myc Oncoproteins Using The AMPAK System", methods IN molecular biology, Vol.10, pp.273 and 281 (The Humana Press, Inc.1992). For example, in a method for detecting RS7 antigen using a dual-determinant ELISA, a minced fine tissue from a biopsy sample is lyophilized and then resuspended in a lysis buffer (100mM NaCl, 50mM Tris-HCl, pH 7.4) containing 1% nonidet-p40(NP40), 0.6. mu.l/ml phthalidase, 0.2mM phenylmethylsulfonyl fluoride, 0.1. mu.g/ml leupeptin and 1mM EDTA at a concentration of 10-20 mg tissue (wet-dry)/500. mu.l solution. The suspension was incubated on ice for 60 minutes and then sonicated for approximately 6 10-second intervals. Insoluble material was removed by centrifugation.

Soluble extracts were added to the wells of the microtiter plates containing the adsorbed RS7 antigen Mab as capture antibody. The captured RS7 antigen was then recognized by a second RS7 antigen MAb that had been conjugated to alkaline phosphatase. The amount of alkaline phosphatase bound, as compared to the RS7 antigen in the extract, can be detected by color measurement using a chromogenic substrate such as p-nitrophenyl phosphate.

Alternatively, a dual determinant ELISA for RS7 antigen can be performed using horseradish peroxidase. Other variations of sample preparation and dual determinant ELISA can be designed by one skilled in the art based on routine experimentation.

In a dual determinant ELISA, the soluble antibody or antibody fragment must bind to an RS7 epitope that is different from the epitope recognized by the capture antibody. For example, the soluble antibody may be the RS7MAb and the capture antibody may be MR 23. Alternatively, the soluble antibody may be MR23 and the capture antibody may be RS7 MAb.

A dual determinant ELISA can be performed to determine whether RS7 antigen is present in a biopsy sample. Alternatively, the assay can be performed to quantify the amount of RS7 antigen present in a clinical sample of bodily fluid. Quantitative analysis can be performed by including dilution of the purified RS7 antigen. Methods for purifying the RS7 antigen are described below.

The RS7MAb and the fragments thereof are also suitable for the preparation of detection kits. The kit may comprise a carrier which is distinguished as being closely received in one or more container means, such as bottles, tubes or the like, each of which comprises a respective element of an immunoassay.

For example, there may be one container means containing the capture antibody immobilized on a solid support and another container means containing the detectably labeled antibody in solution. Another container means may comprise a standard solution containing serial dilutions of RS7 antigen. Standard solutions of RS7 antigen can be used to generate standard curves, where the concentration of RS7 antigen is plotted on the abscissa and the detection signal is on the ordinate. The results obtained from samples containing RS7 antigen can be interpolated from the curves to give the concentration of RS7 antigen in the biological sample.

The RS7 antibodies and fragments thereof of the invention are also useful for detecting the presence of RS7 antigen in sections prepared from immunohistochemical samples. The in situ detection can be used to determine the presence of the RS7 antigen and to determine the distribution of the RS7 antigen in the test tissue. In situ detection can be performed by using detectably-labeled RS7 antigen binding protein for sectioning frozen samples. Studies have shown that the RS7 antigen cannot be preserved in paraffin-embedded sections. Stein et al (1993) as above. The general techniques for in situ detection are well known to those skilled in the art. See, for example, punder, "Cell Marking Techniques and theory Application," mammaliand evalopment: a PRACTICAL APPROACH 113-38Monk (ed.) (IRLPress 1987), and Coligan 5.8.1-5.8.8. See also Stein et al (1989), supra, and Stein et al (1993), supra.

The RS7 antibody and fragments thereof can be detectably labeled with any suitable detection agent, such as radioisotopes, enzymes, fluorescent labels, chemiluminescent labels, bioluminescent labels and paramagnetic labels. Methods for making and detecting the detectably-labeled RS7 antigen binding proteins are well known to those skilled in the art and are described in more detail below.

The label moiety may be a radioisotope that is detected by means of using a gamma counter or scintillation counter or autoradiography. In a preferred embodiment, the diagnostic conjugate is a gamma-, beta-or positron-emitting isotope. The label part of the present specification relates to molecules which will generate a signal under predetermined conditions. Examples of label moieties include radioisotopes, enzymes, fluorescent labels, chemiluminescent labels, bioluminescent labels and paramagnetic labels. As used herein, a diagnostic or therapeutic agent binds to an antibody to produce molecules and atoms of a conjugate that can be used for diagnosis and therapy. Examples of diagnostic or therapeutic agents include drugs, toxins, chelators, dyes, chromophores, boron compounds, and label moieties. Isotopes particularly suitable for the purposes of the present invention are³H，¹³¹I，³¹S，¹⁴C, and preferably¹²⁵1. Examples of other radionuclides are, for example⁹⁰Y，¹¹¹InN，^99mTc，¹⁸⁶Re，¹⁸⁸Re，¹⁷⁷Lu，⁶⁷Cu，²¹²Bi，²¹³Bi, and²¹¹at. Other radionuclides may also be useful as diagnostic and therapeutic agents. Isotopes suitable for diagnostic imaging are typically 25-4,000keV, while therapeutic radionuclides suitable for use are typically 60-700 keVkeV。

The RS7 antibodies and fragments thereof of the invention can also be labeled with fluorescent compounds. The presence of fluorescently labeled Mab is determined by exposing the RS7 antigen binding protein to light of the appropriate wavelength and detecting the fluorescence produced. The fluorescent labeling compound comprises fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluoramine. The fluorescently labeled RS7 antigen binding protein is particularly useful for flow cytometry.

Alternatively, RS7 antigen binding proteins are conjugated to chemiluminescent compounds to provide detectable labels for RS7 antibodies and fragments thereof. The presence of a chemiluminescent-tagged Mab is determined by detecting the luminescence generated in the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazole, acridinium ester salts, and oxalate esters.

Similarly, bioluminescent compounds can be used to label the RS7 antibodies and fragments thereof of the invention. Bioluminescence is a type of bioluminescence found in biological systems in which catalytic proteins increase the efficacy of a chemiluminescent reaction. The presence of bioluminescent proteins is determined by detecting luminescence. Bioluminescent compounds for labeling include luciferin, luciferase and aequorin.

Alternatively, RS7 antibodies and fragments thereof are detectably labeled by linking RS7 antibodies to enzymes. When the RS7 antibody-enzyme conjugate is incubated in the presence of a suitable substrate, the enzyme moiety reacts with the ground substance to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorimetry, or visual means. Examples of enzymes that can be used to detectably label the RS7 antibody include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

RS7 antibodies, fusion proteins, and fragments thereof can also be labeled with paramagnetic ions for in vivo diagnostic purposes. Contrast agents particularly suitable for magnetic resonance imaging include Gd, Mn, Dy or Fe ions. The RS7 antibody and fragments thereof may also be conjugated to ultrasound contrast/enhancing agents. For example, the ultrasound contrast agent is a liposome comprising a humanized RS7 IgG or fragment thereof. More preferably, the ultrasound contrast agent is a gas-filled liposome.

In the relevant vein, the bispecific antibody may be conjugated to a contrast agent. For example, a bispecific antibody may comprise more than one image-enhancing agent for ultrasound imaging. In a preferred embodiment, the contrast agent is a liposome. Preferably, the liposome comprises a bivalent DTPA-peptide covalently bound to the outer face of the liposome. More preferably, the liposomes are gas filled.

Those skilled in the art will be aware of other suitable markers that may be applied in accordance with the present invention. Binding of the marker moiety to the RS7 antibody can be performed using standard methods known in the art. Typical methods related thereto are described in Kennedy et al, clin. 1(1976), Schurs et al, Clin. Chim. acta 81: 1(1977), Shih et al, Int' l J. cancer 46: 1101(1990), Stein et al (1990), supra, and Stein et al (1993), supra. See also, Coligan generally.

The in vitro and in situ detection methods described above can be used to assist in the diagnosis and staging of pathological packages. For example, the methods can be used to detect tumors expressing the RS7 antigen, including tumors of the lung, breast, bladder, ovary, uterus, stomach, and prostate.

In vivo diagnosis

The invention also contemplates the use of the RS7 antibody in vivo diagnostics. Methods of diagnostic imaging using radiolabeled mabs are well known. In immunoscintigraphy, for example, antibodies are labeled with a gamma-emitting isotope and introduced into a patient. The location and distribution of the gamma radioisotope is detected using a gamma camera. See, FOR example, Srivastava (ed.), RADIO LABELEDNONOCLONAL ANTIBODIES FOR IMAGINGAND THERAPY (Plenum Press1988), Chase, "Medical Applications of Aadiosotopes", in REMINGTON' S PHARMACEUTICAL SCIENCES, 18TH Edition, Gennaro et al (eds.), Page 624 & 652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Mnonococcal ANTIBODIES", BIOTECHNOLY & PHMACARY 227-49, Pezzuto et al (eds.) (Chapman & Hall 1993).

For diagnostic imaging, the radioisotope can be conjugated to the RS7 antibody either directly or indirectly using a mediating functional group. Suitable mediator functional groups include chelating agents such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. See, for example, Shih et al, supra, and U.S. Pat. No. 5,057,313.

By choosing a minimum half-life, minimum retention in vivo and the lowest amount of isotope that can allow detection and accurate determination of the isotope in the optimum combination, the radiation dose delivered to the patient is kept as low as possible. Examples of radioisotopes that can bind to RS7 antibody and are suitable for diagnostic imaging include^99mTC and¹¹¹In。

pharmaceutically acceptable excipients

Additional pharmaceutical methods can be used to control the duration of action of the RS7 antibody in therapeutic applications. Controlled release agents can be made by complexing or absorbing RS7 antibody using polymers. For example, biocompatible polymers include poly (ethylene-co-vinyl acetate) matrices and polyanhydride copolymer matrices of stearic acid dimer and sebacic acid. Sherwood et al, Bio/Technology 10: 1446(1992). Sherwood et al, biol technology 10: 1446(1992). The rate of release of RS7 antibody from the matrix depends on the molecular weight of the RS7 antibody, the amount of RS7 antibody within the matrix, and the size of the dispersed particles. Saltzman et al, biophysis.j.55: 163 (1989); sherwood et al, supra. Other solid dosage forms are described in REMINGTON's pharmaceutical SCIENCES, 18TH ed. (1990).

Humanized, chimeric and human RS7 antibodies to be delivered to an individual may comprise only a mAb, an immunoconjugate, an antibody fusion protein or may comprise one or more pharmaceutically acceptable excipients, one or more additional components, or some combination of these.

The immunoconjugates, naked antibodies, fusion proteins and fragments thereof of the invention can be formulated in known ways into pharmaceutically useful compositions, whereby the immunoconjugate or naked antibody and a pharmaceutically suitable excipient are combined in a mixture. Phosphate buffered sterile saline is an example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. See, for example, Ansel et al PHARMACEUTICAL DOSAGE FORMS and drugs DELIVERYSYSTEMS, 5TH Edition (Lea & Febiger 1990), and GENNARO (ed.), REMINGTON' S PHARMACEUTICAL SCIENCES, 18TH Edition (MackPublishing Company1990), and revisions thereof.

The immunoconjugates or naked antibodies of the invention can be formulated for intravenous administration by, for example, bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an additional preservative. The compositions may take such forms as suspensions, solutions or emulsions, whether oily or aqueous, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., pyrogen-free sterile water, before use.

Additional pharmaceutical methods can be used to control the duration of action of the therapeutic or diagnostic conjugate or naked antibody. Controlled release agents can be made by complexing or absorbing the immunoconjugate or naked antibody with a polymer. For example, biocompatible polymers include poly (ethylene-co-vinyl acetate) matrices and polyanhydride copolymer matrices of stearic acid dimer and sebacic acid. Sherwood et al, Bio/Technology 10: 1446(1992). The rate of release of the immunoconjugate or antibody from the matrix depends on the molecular weight of the immunoconjugate or antibody, the immunoconjugate within the matrix, the amount of antibody, and the size of the dispersed particles. Saltzman et al, biophysis.j.55: 163 (1989); sherwood et al, supra. Other solid DOSAGE FORMS are described in Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND PHARMACEUTICALs DELIVERY SYSTEMS, 5THEDITION (Lea & Febiger 1990) AND Gennaro (ed.), REMINGTON' SPHARMACEMENT SCIENCES, 18TH Edition (Mack Publishing Company1990) AND revisions thereof.

Immunoconjugates, antibody fusion proteins, naked antibodies and fragments thereof can also be administered to a mammal subcutaneously or even by other parenteral routes. In a preferred embodiment, the anti-EGP-1 antibody or fragment thereof is administered at a dose of 10-2000 mg protein per dose. Moreover, administration can be by continuous infusion, or single or multiple bolus infusions. In general, the dose of the immunoconjugate, fusion protein or naked antibody administered to a human varies depending on factors such as the age, weight, height, sex, general medical condition and past medical history of the patient. Typically, it is desirable to provide the immunoconjugate, antibody fusion protein, or naked antibody to the recipient at a dose of about 1mg/kg to 20mg/kg in a single intravenous infusion, although lower or higher doses may also be administered as circumstances require. The dose may be administered repeatedly as needed, for example once a week for 4 to 10 weeks, preferably once a week for 8 weeks, more preferably once a week for 4 weeks. It may also be given less frequently, such as once every other week for months. The dosage can be administered by a variety of parenteral routes, and the dosage and regimen can be suitably adjusted.

The RS7 antibody of the invention can be formulated in a known manner into a pharmaceutically useful composition, whereby the RS7 antibody and a pharmaceutically suitable excipient are combined in a mixture. If administration of the composition is tolerated by the patient to be treated, the combination is referred to as a "pharmaceutically acceptable carrier". Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. See, for example, REMINGTON' SPHARMACEMENT SCIENCES, 18TH Ed (1990).

For therapeutic purposes, the immunoconjugate, fusion protein, or naked antibody is administered to the mammal in a therapeutically effective amount. Suitable subjects for which the invention is directed are typically humans, but non-human animal subjects are also contemplated. An antibody preparation is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient mammal.

Various modifications and alterations to the compositions and methods of this invention will be apparent to those skilled in the art. It is, therefore, to be understood that the invention is intended to cover such modifications and changes as fall within the scope of the appended claims and equivalents thereof.

All publications, patents and patent applications cited above are herein incorporated by reference in their entirety to the same extent as if each had been individually incorporated by reference.

The following examples are illustrative of embodiments of the present invention and are not intended to limit the scope of the claims in any way.

Example 1 construction of chimeric RS7 antibodies

Molecular cloning of RS7V kappa and VH genes

Total cytoplasmic RNA and mRNA were prepared from RS 7-producing hybridoma cells. The genes encoding the V.kappa.and VH sequences were cloned using RT-PCR and 5' RACE and then sequenced by DNA sequencing. Multiple clones were sequenced to eliminate possible errors caused by PCR reactions. Sequence analysis showed the presence of two vk (#1 and #23) and one VH (RS7VH) transcript. Two chimeric ab (cabs) containing human constant regions were generated in combination with each potential murine VK and VH and then expressed in Sp2/0 cells by transfection. Clones producing cabs were identified by screening cell culture supernatants of transfected cell clones by ELISA. Positive clones were expanded and cabs were purified from cell culture supernatants. Ag-binding assays showed that cabs consisted of V κ #23 and VH, and cabs-V κ #23 bound to microwells coated with ME180, a crude membrane fraction of human cervical cancer cells (ATCC, Rockville, MD) (fig. 1). cAb associates V κ #1 and VH, and cAb-V κ #1 did not show binding to Ag-coated wells.

Thus, the immunoreactive cabs (containing V κ 23) were designated cRS 7. Cloned murine VH and functional vk (#23) sequence products as PCR products were designated RS7V κ (fig. 2A) and RS7VH (fig. 2B), respectively.

RS7 Ab binding Activity detection

A competitive ELISA binding assay was used to evaluate the binding affinity of the engineered cRS 7. Briefly, a constant amount of biotinylated murine RS7 was mixed with varying concentrations (0.01-100. mu.g/ml) of detection Ab (RS7 or cRS7), added to Ag-coated microwells, and incubated at room temperature for 1 hour. After washing, HRP-conjugated streptavidin was added and then incubated for 1 hour at room temperature. After adding the solution containing 4mM o-phenylenediamine dihydrochloride and 0.04% H₂O₂By reading the OD₄₉₀But shows the amount of HRP-conjugated streptavidin bound to Ag-bound biotinylated RS 7. By this type of competitive Ag-binding assay, it was shown that the competition of cRS7 and murine RS7 for binding of biotinylated murine RS7 to antigen-coated wells was identical, thus confirming the reliability of the obtained vk and VH sequences (fig. 1).

Example 2 construction method of hRS7 antibody

Sequence design of hRS7V Gene

By studying the human vk and VH sequences in the Kabat database, FRs for RS7V k and VH were found to show a high degree of sequence identity to human SA-1A' c1 vk and RF-TS3 VH, respectively. One exception is FR4 of RS7VH, which shows the highest identity with the NEWM VH. Thus, the human SA-1A' c1 framework sequence was used as a scaffold for grafting the CDRs of RS7V K (FIG. 3A), and RF-TS3 and NEWM framework sequences were used in combination for RS7V_H(FIG. 4). There are a large number of amino acid changes outside each chain CDR region when compared to the starting human antibody framework. Some of the amino acid residues flanking potential CDRs in murine FR remain based on the amino acid sequences described by Qu, z., Losman, m.j., elissen, k.c., Hansen, h.j., golden berg, d.m., and Leung, s.o. (1999) humanization of mmu31, alpha-fetoprotein-specific. Clin.cancer Res.5, 3095s-3100s in hRS7 Fv reconstructed under the previously established guidelines. These residues are S20, D60, V85, and a100 for RS7V κ and K38, K46 for RS7VH, a78 and F91 (fig. 3A and 3B).

Construction of hRS7V sequence

Leung et al Leung, s.o., Shevitz, j., Pellegrini, m.c., Dion, a.s., Shih, l.b., Goldenberg, d.m., and Hansen, H.J. (1994) polymerization of LL 2a rapid interacting antibody specific for Bcell lymphama. hybridoma, 13: 469-476) discloses a modification strategy that is used to construct VL and VH genes designed for hRS7 using long oligonucleotide synthesis and PCR as shown in figure 4. To construct the hRS7VH region, two long oligonucleotides, hRS7VHA (176-MER) and hRS7VHB (168-MER), were synthesized on an automated DNA synthesizer (Applied biosystems).

The hRS7VHA represents 23-198 nt of the hRS7VH region

5’-GGTCTGAGTT GAAGAAGCCT GGGGCCTCAG TGAAGGTTTC

CTGCAAGGCT TCTGGATACA CCTTCACAAA CTATGGAATG AACTGGGTGA

AGCAGGCCCC TGGACAAGGG CTTAAATGGA TGGGCTGGAT AAACACCTAC

ACTGGAGAGC CAACATATAC TGATGACTTC AAGGGA-3’

The hRS7VHB represents the minus strand of the hRS7VH region complementary to 174-340 nt.

5’-ACCCTTGGCC CCAGACATCG AAGTACCAGT AGCTACTACC

GAACCCCCCT CTTGCACAGA AATACACGGC AGTGTCGTCA GCCTTTAGGC

TGCTGATCTG GAGATATGCC GTGCTGACAG AGGTGTCCAA GGAGAAGGCA

AACCGTCCCT TGAAGTCATC AGTATATG-3’

The 3' terminal sequences (23nt residues) of hRS7VHA and B were complementary to each other. Under the PCR conditions, the hRS7VHA and B3' -ends anneal to form a short double-stranded DNA flanked by the remaining portions of the long oligonucleotide. Each annealed end serves as a primer for transcription of the single-stranded DNA, resulting in 23 to 340nt of double-stranded DNA comprising hRS7 VH. This DNA was further amplified in the presence of two short oligonucleotides, hRS7VHBACK and hRS7VHFOR to form full-length hRS7 VH.

hRS7VHBACK 5’-GTGGTGCTGC AGCAATCTGG GTCTGAGTTG

AAGAAGCC-3’

hRS7VHFOR 5’-TGAGGAGACG GTGACCAGGG ACCCTTGGCC

CCAGACAT-3’

10 μ l of 10 XPCR buffer (500mM KCl, 100mM Tris. HCl buffer, pH 8.3, 15mM MgCl)₂) Mu. mol of hLL1VHBACK and hLL1VHFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Ct) amplified the minimal amount of hRS7VHA and B (determined empirically). The reaction mixture was subjected to 3 cycles of PCR reaction: denaturation at 94 ℃ for 1 min, annealing at 45 ℃ for 1 min, and polymerization at 72 ℃ for 1.5 min, followed by 27 cycles of PCR: denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min, and polymerization at 72 ℃ for 1 min. The double-stranded PCR-amplified product of hRS7VH was gel-purified, restricted with PstI and BstEII and then cloned into the heavy chain staging vector, the complementary PstI/BstEII site of VHpBS 2.

To construct full-length DNA of humanized V.kappa.sequences, hRS7VKA (156-mer) and hRS7VKB (155-mer) were synthesized as described above. hRS7VKA and B were amplified by the short oligonucleotides hRS7 VKPACK and hRS7VKFOR described above.

hRS7VKA represents 20-175 nt of the hHRS7V kappa region.

5’-CTCCATCCTC CCTGTCTGCA TCTGTAGGAG ACAGAGTCAG CATCACCTGC

AAGGCCAGTC AGGATGTGAG TATTGCTGTA GCCTGGTATC AGCAGAAACC

AGGGAAAGCC CCTAAGCTCC TGATCTACTC GGCATCCTAC CGGTACACTG

GAGTCC-3’

hRS7VKB represents the minus strand of hRS7V kappa region, which is complementary to 155-320 nt.

5’-CCTTGGTCCC AGCACCGAAC GTGAGCGGAG TAATATAATG

TTGCTGACAG TAATAAACTG CAAAATCTTC AGGTTGCAGA CTGCTGATGG

TGAGAGTGAA ATCTGTCCCA GATCCACTGC CACTGAACCT ATCAGGGACT

CCAGTGTACC GGTAG-3’

hRS7VKBACK 5’-GACATTCAGC TGACCCAGTC TCCATCCTCC

CTGTCTG-3’

hRS7VKFOR 5’-ACGTTAGATC TCCACCTTGG TCCCAGCACC G-3’

The double-stranded PCR-amplified product of hRS7V κ was gel-purified, restriction-digested with PvuII and BGLIII and then cloned into the light chain staging vector, the PvuI/BcII complement site of VKPCR 2. The final expression vector was constructed by sequential subcloning of XBAI-BAMHI and XhoI/BamHI fragments of HRS7V kappa and VH, respectively, into pdHL2 as described above.

Transfection and expression of hRS7 antibodies

Approximately 30. mu.g of the expression vector for hRS7 was linearized by SalI digestion and then transfected into SP2/0-AGL4 cells using electroporation (450V and 25 PF). Transfected cells were plated in 96-well plates for 2 days and drug resistance was selected by adding MTX to a final concentration of 0.025. mu.M.

MTX-resistant colonies appeared in the wells within 2-3 weeks. Supernatants from selected clones surviving for screening for human Ab secretion were tested by ELISA assay. Briefly, 100. mu.l of the supernatant sample was added to the sample with GAH-IgG, F (ab')₂Fragment-specific Ab pre-coated ELISA microtiter plates and then incubated for 1 hour. The plate was washed three times with wash buffer (PBS containing 0.05% polysorbate 20) to remove unbound protein. HRP-conjugated GAH-IgG, Fc fragment-specific Ab was added to the wells.

After incubation for 1h, the plates were washed. After adding the mixture containing 4mM OPD and 0.04% H₂O₂After the substrate solution is read by₄₉₀And shows bound HRP-binding. Positive cell clones were expanded and hRS7 IgG was purified from cell culture supernatants by protein a column affinity chromatography.

Binding Activity of humanized RS7 antibody

ELISA competitive binding assays were used to evaluate immunoreactivity of hRS7 using ME180 cell membrane extract coated plates as described (Stein et al, int. J. cancer 55: 938-946 (1993)). The ME180 cell membrane fraction was prepared by sonication and centrifugation. 96-well flat-bottom PVC plates were coated with crude membrane extract by centrifugation and then fixed with 0.1% glutaraldehyde. A constant amount of biotinylated murine RS7 was mixed with varying concentrations of mRS7, cRS7 or hRS7, then added to the coated microwells, followed by incubation at room temperature for 1 hour. After washing, HRP-conjugated streptavidin was added, followed by incubation at room temperature for 1 hour. After adding the solution containing 4mM o-phenylenediamine dihydrochloride and 0.04% H₂O₂After the substrate solution is read by₄₉₀While the amount of HRP-conjugated streptavidin bound to the membrane-bound biotinylated mRS7 is shown. As shown by the competition assay in fig. 6, hRS7 IgG showed comparable binding activity to mRS7 and cRS7, confirming that the binding affinity of RS7 was preserved in humanization.

Example 3 radioiodination of humanized RS7 Using residual marker

The residual moiety (IMP-R4, IMP-R5 or IMP-R8) was radioiodinated and coupled to disulfide-reduced hRS7 as described elsewhere (Govindan SV, et al, Bioeonjoute chem.1999; 10: 231-240). See fig. 9. In residual radioiodination, use¹²⁵I, preparation of¹²⁵I-IMP-Rx-hRS7 where x is 4,5 or 8), 87.1% (3.38mCi/mg), 34.3% (0) of the total yield and specific activity (in brackets) were obtained using IMP-R4, IMP-R5 and IMP-R8, respectively.97mCi/mg), and 76.6% (2.93 mCi/mg). At a large scale¹³¹In I marking, use¹³¹I-IMP-R4 body, the following results were obtained. Use of 20.4mCi¹³¹I, 35.7nmol IMP-R4 and 3.22mg DTT-reduced RS7, gave 60% overall yield (3.80 mCi/mg). Use of 30.3mCi¹³¹I, IMP-R4 and reduced hRS7 Another run gave 69.7% yield (3.88 mCi/mg).

13.97mCi¹³¹The third run of I gave a degree of incorporation of 71.8% (4.42 mCi/mg). With 13.6mCi¹³¹I and a non-specific humanized antibody, hLL 2-labeled¹³¹I-IMP-R4 gave a 64.4% yield (3.67 mCi/mg).

Example 4 preclinical testing in animal models of breast cancer

For tumor targeting experiments, approximately 2.3X 10 were injected subcutaneously⁷The cultured MDA-MB-468 cells propagate tumors in 5-8 weeks old female nude mice, and after 1 month, when the tumor size reaches-0.1-0.2 cm³Animals were used. Mice were injected intravenously with 10 μ Ci¹²⁵I-[IMP-Rx]-hRS7 where x is 4,5 or 8, and 20-25 μ Ci¹³¹I-Mab (CT method). Therefore, each experiment was conducted¹²⁵I/¹³¹I paired labeling experiment. Biodistribution in various organs and blood was measured at the indicated times and then expressed as% injected dose/g. In determining¹²⁵Correction when biodistribution is observed¹³¹I backscatter into¹²⁵The amount of I window.

For therapeutic studies, tumor growth patterns in various forms were studied in order to determine the best method for stable tumor growth. It was concluded that the method for targeted experiments after about 8-weeks of tumor growth was optimal and that 30-50% of the animals could be used depending on the tumor growth pattern. Intravenous injection of animals bearing tumors is the test agent for therapeutic studies¹³¹I-IMPR4-hRS7, and a direct radioiodinated substance¹³¹I-hRS7 was used for comparison.

Baseline body weights were compared to weekly body weights and tumor volumes. When the tumor reaches 3cm³Time-to-death animals. All animal experiments were performed according to the IACUC-approved method.

Biodistribution in animals

Use of a dual-labeled hRS7 preparation in growing tumors of NIH Swiss nude mice: (¹²⁵I-IMP-Rx-hRS7 where x is 4,5 or 8, each reagent being directly labelled¹³¹I-hRS7 mix) were performed. Tables 1A, 1B and 1C describe that specific biodistributions show very good performance using residual markers. For example¹²⁵I-IMP-IMP-R4-RS7，¹²⁵I-IMP-R5-hRS7 and¹²⁵the% injected dose of I-IMP-R8-hRS7 on day 7/g tumor was 41.6. + -. 3.0%, 32.2. + -. 11.6% and 24.7. + -. 8.5%, respectively, compared to that for direct labeling¹³¹I-HRS7 was found to be 5.9. + -. 0.9%, 6.2. + -. 2.1% and 6.7. + -. 2.3% at the same time points in each double-labelled experiment. At the same time point, the tumor-to-non-tumor ratio is¹²⁵I-IMP-R4-hRS71.7-7.6 times,¹²⁵I-IMP-R5-hRS71.7-6.0 times and¹²⁵I-IMP-R8-hRS72.0-4.8 times higher than that of I-IMP-R8-hRS72.0¹³¹I-hRS7 (data not shown).

TABLE 1. use ¹²⁵ I-IMP-R (R4 or R5 or R8) and ¹³¹ I-hRST (CT method) double-labeling Humanized RS7 in NIH Swiss nude mice bearing MDA-MB-468 tumor xenografts Biodistribution in

Table 1A: ¹²⁵ I-IMP-R4-hRS7 pair ¹³¹ I-hRS7(CT)

Table 1B: ¹²⁵ I-IMP-R5-hRS7 pair ¹³¹ I-hRS7(CT method)

Table 1C: ¹²⁵ I-IMP-R8-hRS7 pair ¹³¹ I-hRS7(CT method)

Dosimetric calculation, use according to biodistribution¹²⁵I replacement¹³¹I, carried out using the methods of Siegel, JA and Stabin, MG (Journal of Nuclear Medicine 1994; 35: 152-. Table-2 compares the residual and conventional radioiodination, while figure 10 graphically depicts the data. All residual agents were found to perform best in the manner of delivered tumor dose and tumor-to-non-tumor ratio; selected in view of beneficial radiochemical production and specific activity obtainable for the same agent¹³¹I-IMP-R4-HRS7 was used in the treatment experiments.

Table 2: in the MDA-MB-468 tumor model due to Radiation dose calculated by varying radioiodinated hRS7

Treatment of MDA-MB-468 human breast cancer xenografts in nude mice

Maximum Tolerated Dose (MTD):from the dose determination data (table-2, group 1),¹³¹I-IMP-R4-hRS7 and¹³¹the amount of mCi of I-hRS7 was calculated to be 0.231mCi and 0.285mCi, respectively, at a radiation dose of 1500cGy (estimated MTD) to blood. Experimental determination of MTD was performed in Swiss nude mice with increasing doses of each agent. For the¹³¹I-IMP-R4-HRS7, administered to groups of animals 200, 225, 250, 275, 300 and 325 μ Ci; 1 of 5 animals in the 250 μ Ci dose group died after 4 months, while 3 of 4 animals in the 300 μ Ci dose group died between 2 weeks and 4 weeks. Although it was unexpected that animals in both 275 and 325 μ Ci dose groups survived at week 5, we summarized the MTD between the 231 μ Ci (calculated from dose-determination data) and 250 μ Ci dose-administration groups. For the¹³¹I-HRS7 (radioiodinated based on "CT"), groups of animals injected with 250, 280, 310, 340, 370 and 400 μ Ci; between 2 weeks and 3 weeks, 6 of 6 animals in the 340. mu. Ci dose group, 3 of 6 animals in the 370. mu. Ci dose group, and 4 of 4 animals in the 400. mu. Ci dose group died. Based on these, the MTD was set to 280 to 310. mu. Ci.

Therapeutic study-1

For the purpose of¹³¹Potency and potency of I-IMP-R4-hRS7¹³¹For the first therapeutic experiment, where the efficacy of I-hRS7(CT method) was compared, each agent was used at-70% of its maximum tolerated dose. A single dose of 175 μ Ci residual reagent showed a potency significantly higher than that of a conventional radioiodine of 200 μ Ci. In this experiment including untreated controls, 10 or 11 animals were used per group, and all three groups were randomized in order to make their starting tumor sizes very similar. The mean tumor volumes of the three groups before treatment (day 2) were 0.312 ± 0.181, 0.308 ± 0.203 and 0.303 ± 0.212.

In this experiment, the transition data on day 49 is shown below FIG. 11. The upper panel in FIG. 11 shows the tumor volume (CM) of each animal in each group³) While the lower panel shows the mean tumor volume in both formats. Three animals died in the untreated group. Tumor growth control in the residual marker group is significantly better than in the traditional marker and untreated groups, e.g., on Mean Tumor Volume (MTV) up toArea under the curve (AUC) at day 49 as determined by student-t test. At 49 days, due to use¹³¹Significant differences in AUC (p-value) for MTV upon I-IMP-R4-hRS7 treatment are shown, with p-values for the difference in tumor volume before treatment (day 2) given in parentheses, as follows. For untreated: 0.05 (0.78); for the¹³¹I-hRS7 (CT): 0.03 (0.98); for the¹³¹I-hRS7(CT) vs untreated: 0.14(0.81). At day 49, there was a sustained dispersion of mean tumor volume between the regular and residual radioiodine groups, which resulted in a sustained decrease. 8-week after treatment, in use¹³¹In the group treated with I-IMP-R4-hRS7, 5 of 11 mice were in complete remission with MTV of 20% of the initial value. Untreated and¹³¹I-hRS 7-treated mice had MTV at 8 weeks of 280% and 163% of each initial value,¹³¹complete remission was obtained in 1 of 11 mice in the I-hRS7 group.

The treatment is well tolerated. The mean body weight of the IMP-R4 group was 21.93. + -. 2.03 on day 2 and 23.68. + -. 1.81 on day 49; for the "CT" group, the mean body weights were 21.77. + -. 2.21 and 23.90. + -. 2.64 on days 2 and 49, respectively. Toxicity in bone marrow of the treated groups, as determined by blood cell counts, is shown in FIG. 12. Briefly: for use of¹³¹I-IMP-R4-HRS7 reached the lowest values of 34%, 7% and 61% of the control levels, respectively, for WBC, lymphocyte and neutrophil counts 1 week after dosing. At week 5, these returned to 74%, 58% and 92% of the control levels, respectively, and remained at 45%, 36% and 51% of the control levels on day 49; to for¹³¹I-hRS7 (CT): the WBC, lymphocyte and neutrophil counts reached the lowest values of 41%, 13% and 67%, respectively, of the control level 1 week after administration of the agent. At week 5, these returned to 85%, 67% and 103% of the control levels, respectively, and remained 42%, 32% and 49% of the control levels on day 49.

Therapeutic study-2

Use in MDA-MB-468 tumor model¹³¹RAIT specificity of I-IMP-R4-hRS7

Will be provided with¹³¹Potency and non-specific use of I-IMP-R4-hRS7¹³¹I-IMP-R4 labeled control humanized antibody, hLL2 (anti-CD 22MAb), was compared for efficacy. In this experiment, 175. mu. Ci of each reagent was administered. This represents¹³¹I-IMP-R4-hRS7 maximum tolerated dose 70%. In this experiment including untreated controls, 7-8 animals per group were used, as in treatment experiment 1, and were randomly grouped with reference to the initial tumor volume distribution. FIG. 13, showing the relative Mean Tumor Volume (MTV) of the three groups (MTV before treatment: 100), shows specific growth regulation.

Example 5 treatment of breast cancer patients with Y-90 humanized RS7mAb and naked humanized RS7 mAb:

a woman, 56 years old, has a history of recurrence of breast cancer, manifested as metastasis to the cervical lymph nodes and left lung. The patient relapsed twice after chemotherapy and hormone treatment. The patient then received two therapeutic injections (2 weeks apart) of Y-90-conjugated humanized RS7mAb i.v., at a dose of 20mCi Y-90 in 100mg of antibody protein. Four weeks after treatment, the patient had a reduction in white blood cell and platelet counts of about 50%, but recovered at 9 weeks after treatment. At a further tumor classification of 12 weeks after treatment, a 30% reduction in lung and lymph node metastasis was detected by computer tomography. Thereafter, the patient received 4 weekly infusions (each over 3 hours) of well tolerated naked humanized RS7 without any adverse effects on their blood count or blood chemistry except for some brief rigor and chills. The naked antibody per infusion was 400mg/m². After about 8 weeks, re-tumor classification via computed tomography showed an additional reduction of about 20% in detectable lesions. The patient's disease appeared stable (i.e., showed no additional or progressive growth) after 3 months of follow-up testing.

Sequence listing

<110>IMMUNOMEDICS，INC.

<120> RS7 antibody

<130>018733/1164

<140>PCT/GB02/00885

<141>2003-03-03

<150>60/360,229

<151>2002-03-01

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Asp Ile Gln Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

gac agg gtc agc atc acc tgc aag gcc agt cag gat gtg agt att gct 96

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

gta gcc tgg tat caa cag aaa cca gga caa tct cct aaa cta ctg att 144

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

tac tcg gca tcc tac cgg tac act gga gtc cct gat cgc ttc act ggc 192

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

agt gga tct ggg acg gat ttc act ttc acc atc agc agt gtg cag gct 240

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

gaa gac ctg gca gtt tat tac tgt cag caa cat tat att act ccg ctc 288

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

acg ttc ggt gct ggg acc aag ctg gag ctg aaa cgg 324

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

100 105

<210>2

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Asp Ile Gln Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

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

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gtg aag ctg cag gag tca gga cct gag ctg aag aag cct gga gag aca 48

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

1 5 10 15

gtc aag atc tcc tgc aag gct tct gga tat acc ttc aca aac tat gga 96

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

20 25 30

atg aac tgg gtg aag cag gct cca gga aag ggt tta aag tgg atg ggc 144

Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly

35 40 45

tgg ata aac acc tac act gga gag cca aca tat act gat gac ttc aag 192

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

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gga cgg ttt gcc ttc tct ttg gaa acc tct gcc acc act gcc tat ttg 240

Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Thr Ala Tyr Leu

65 70 75 80

cag atc aac aac ctc aaa agt gag gac atg gct aca tat ttc tgt gca 288

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

85 90 95

aga ggg ggg tte ggt agt agc tac tgg tac ttc gat gtc tgg ggc caa 336

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

100 105 110

ggg acc acg gtc acc gtc tcc tca 360

Gly Thr Thr Val Thr Val Ser Ser

115 120

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

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

20 25 30

Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly

35 40 45

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

50 55 60

Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Thr Ala Tyr Leu

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

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

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

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

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Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

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Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe Thr

50 55 60

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

65 70 75 80

Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala

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Arg Glu Asp Ser Asn Gly Tyr Lys Ile Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser

115

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gac atc cag ctg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48

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

1 5 10 15

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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

tac tcg gca tcc tac cgg tac act gga gtc cct gat agg ttc agt ggc 192

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

agt gga tct ggg aca gat ttc act ctc acc atc agc agt ctg caa cct 240

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

65 70 75 80

gaa gat ttt gca gtt tat tac tgt cag caa cat tat att act ccg ctc 288

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

acg ttc ggt gct ggg acc aag gtg gag atc aaa cgt 324

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg

100 105

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<223> description of artificial sequences: humanized hRS7Vk amino acid sequence

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

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

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

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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg

100 105

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<223> description of artificial sequences: humanized hRS7VH sequences

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cag gtc caa ctg cag caa tct ggg tct gag ttg aag aag cct ggg gcc 48

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

1 5 10 15

tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc aca aac tat 96

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

20 25 30

gga atg aac tgg gtg aag cag gcc cct gga caa ggg ctt aaa tgg atg 144

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

ggc tgg ata aac acc tac act gga gag cca aca tat act gat gac ttc 192

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

50 55 60

aag gga cgg ttt gcc ttc tcc ttg gac acc tct gtc agc acg gca tat 240

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

65 70 75 80

ctc cag atc agc agc cta aag gct gac gac act gcc gtg tat ttc tgt 288

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

gca aga ggg ggg ttc ggt agt agc tac tgg tac ttc gat gtc tgg ggc 336

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

100 105 110

caa ggg tcc ctg gtc acc gtc tcc tca 363

Gln Gly Ser Leu Val Thr Val Ser Ser

115 120

<210>10

<211>121

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: humanized hRS7VH amino acid sequence

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

1 5 10 15

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

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

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

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser

115 120

<210>11

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<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: humanized hRS7k sequences

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atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

gtc cac tcc gac atc cag ctg acc cag tct cca tcc tcc ctg tct gca 96

Val His Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala

20 25 30

tct gta gga gac aga gtc agc atc acc tgc aag gcc agt cag gat gtg 144

Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val

35 40 45

agt att gct gta gcc tgg tat cag cag aaa cca ggg aaa gcc cct aag 192

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

50 55 60

ctc ctg atc tac tcg gca tcc tac cgg tac act gga gtc cct gat agg 240

Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg

65 70 75 80

ttc agt ggc agt gga tct ggg aca gat ttc act ctc acc atc agc agt 288

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

ctg caa cct gaa gat ttt gca gtt tat tac tgt cag caa cat tat att 336

Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile

100 105 110

act ccg ctc acg ttc ggt gct ggg acc aag gtg gag atc aaa cgt act 384

Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr

115 120 125

gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg 432

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc 480

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

145 150 155 160

aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt 528

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc acc tac 576

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac 624

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc 672

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

aca aag agc ttc aac agg gga gag tgt tag 702

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210>12

<211>233

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: humanized hRS7k amino acid sequence

<400>12

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala

20 25 30

Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val

35 40 45

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

50 55 60

Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile

100 105 110

Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

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

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210>13

<211>1410

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: humanized hRS7H sequences

<220>

<221>CDS

<222>(1)..(1407)

<400>13

atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

gtc cac tcc gtc caa ctg cag caa tct ggg tct gag ttg aag aag cct 96

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

20 25 30

ggg gcc tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc aca 144

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

35 40 45

aac tat gga atg aac tgg gtg aag cag gcc cct gga caa ggg ctt aaa 192

Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys

50 55 60

tgg atg ggc tgg ata aac acc tac act gga gag cca aca tat act gat 240

Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp

65 70 75 80

gac ttc aag gga cgg ttt gcc ttc tcc ttg gac acc tct gtc agc acg 288

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

85 90 95

gca tat ctc cag atc agc agc cta aag gct gac gac act gcc gtg tat 336

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

100 105 110

ttc tgt gca aga ggg ggg ttc ggt agt agc tac tgg tac ttc gat gtc 384

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

115 120 125

tgg ggc caa ggg tcc ctg gtc acc gtc tcc tca gcc tcc acc aag ggc 432

Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480

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

145 150 155 160

aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa 720

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

225 230 235 240

tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc 768

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816

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

260 265 270

ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1056

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1104

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

355 360 365

cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag 1152

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

370 375 380

gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200

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

385 390 395 400

gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1248

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc aag ctc 1296

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

420 425 430

acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

ctg tct ccg ggt aaa tga 1410

Leu Ser Pro Gly Lys

465

<210>14

<211>469

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: humanized hRS7H amino acid sequence

<400>14

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

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

20 25 30

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

35 40 45

Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys

50 55 60

Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

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

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

225 230 235 240

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

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

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

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

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

370 375 380

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

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly Lys

465

<210>15

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>15

Lys Ala Ser Gln Asp Val Ser Ile Ala Val Ala

1 5 10

<210>16

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>16

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210>17

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>17

Gln Gln His Tyr Ile Thr Pro Leu Thr

1 5

<210>18

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>18

Asn Tyr Gly Met Asn

1 5

<210>19

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>19

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

1 5 10 15

Gly

<210>20

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>20

Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val

1 5 10

<210>21

<211>176

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>21

ggtctgagtt gaagaagcct ggggcctcag tgaaggtttc ctgcaaggct tctggataca 60

ccttcacaaa ctatggaatg aactgggtga agcaggcccc tggacaaggg cttaaatgga 120

tgggctggat aaacacctac actggagagc caacatatac tgatgacttc aaggga 176

<210>22

<211>168

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>22

acccttggcc ccagacatcg aagtaccagt agctactacc gaacccccct cttgcacaga 60

aatacacggc agtgtcgtca gcctttaggc tgctgatctg gagatatgcc gtgctgacag 120

aggtgtccaa ggagaaggca aaccgtccct tgaagtcatc agtatatg 168

<210>23

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>23

gtggtgctgc agcaatctgg gtctgagttg aagaagcc 38

<210>24

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>24

tgaggagacg gtgaccaggg acccttggcc ccagacat 38

<210>25

<211>156

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>25

ctccatcctc cctgtctgca tctgtaggag acagagtcag catcacctgc aaggccagtc 60

aggatgtgag tattgctgta gcctggtatc agcagaaacc agggaaagcc cctaagctcc 120

tgatctactc ggcatcctac cggtacactg gagtcc 156

<210>26

<211>155

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>26

ccttggtccc agcaccgaac gtgagcggag taatataatg ttgctgacag taataaactg 60

caaaatcttc aggttgcaga ctgctgatgg tgagagtgaa atctgtccca gatccactgc 120

cactgaacct atcagggact ccagtgtacc ggtag 155

<210>27

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>27

gacattcagc tgacccagtc tccatcctcc ctgtctg 37

<210>28

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>28

acgttagatc tccaccttgg tcccagcacc g 31

1

15

Claims (25)

1. Use of a chimeric, humanized or human anti-EGP-1 antibody or fragment thereof, wherein the antibody or fragment is a naked antibody or fragment thereof or conjugated to a therapeutic agent other than an Rnase, for the preparation of a medicament for the treatment of a disease in which diseased cells express EGP-1 antigen.

2. The use of claim 1, wherein said anti-EGP-1 antibody or fragment thereof is a naked antibody or fragment thereof.

3. The use of claim 1, wherein the anti-EGP-1 antibody or fragment thereof is conjugated to a therapeutic agent other than Rnase.

4. The use of claim 3, wherein the therapeutic agent is selected from the group consisting of a drug, a toxin, a radionuclide, an immunomodulator, a hormone, an enzyme, a growth factor and an antisense oligonucleotide.

5. The use of claim 4, wherein the toxin is a Pseudomonas endotoxin, ricin, abrin, DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin or Pseudomonas exotoxin.

6. The use of claim 4, wherein the immunomodulator is a cytokine, stem cell growth factor, lymphotoxin, Tumor Necrosis Factor (TNF), hematopoietic factor, interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, colony stimulating factor, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon- α, interferon- β, interferon- γ, stem cell growth factor designated "S1 factor", erythropoietin, thrombopoietin or a combination thereof.

7. The use of claim 3, wherein the therapeutic agent has a pharmacological property selected from the group consisting of antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic, alkaloid, antibiotic agents, and combinations thereof.

8. The use of claim 3, wherein the therapeutic agent is selected from the group consisting of nitrogen mustards, aziridine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, tyrosine kinase inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical hormone inhibitors, antagonists, endothelial somatostatins, paclitaxel, camptothecins, doxorubicin analogs, and combinations thereof.

9. The use of claim 4, wherein the radionuclide is selected from¹³¹I、¹²³I、¹²⁴I、⁸⁶Y、⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、^99mTc、^94mTc、⁹⁰Y、¹¹¹In、¹²⁵I、¹⁸⁶Re、¹⁸⁸Re、¹⁸⁹Re、¹⁷⁷Lu、⁶⁷Cu、²¹²Bi、²¹³Bi、²¹¹At、¹⁹⁸Au、²²⁴Ac、¹²⁶I、¹³³I、⁷⁷Br、^113mIn、⁹⁵Ru、⁹⁷Ru、¹⁰³Ru、¹⁰⁵Ru、¹⁰⁷Hg、²⁰³Hg、^121mTe、^125mTe、¹⁶⁵Tm、¹⁶⁷Tm、¹⁶⁸Ag、¹¹¹Ag、¹⁹⁷Pt、¹⁰⁹Pd、³²P、³³P、⁴⁷Sc、¹⁵³Sm、¹⁰⁵Rh、¹⁴²Pr、¹⁴³Pr、¹⁶¹Tb、¹⁶⁶Ho、¹⁹⁹Au、⁵⁷Co、⁵⁸Co、⁵¹Cr、⁵⁹Fe、¹⁸F、⁷⁵Se、²⁰¹Tl、²²⁵Ac、⁷⁶Br、⁸⁶Y、¹⁶⁹Yb、¹⁶⁶Dy、²¹²Pb and²²³Ra。

10. the use of claim 1, wherein the anti-EGP-1 antibody or fragment thereof is part of a fusion protein.

11. The use of claim 10, wherein the fusion protein comprises a second antibody or fragment thereof or a therapeutic protein or peptide.

12. The use of claim 10, wherein the second antibody or fragment thereof is conjugated to a targeting construct comprising one or more haptens.

13. The use of claim 11, wherein the second antibody or fragment thereof binds to a tumor associated antigen other than EGP-1.

14. The use of claim 11, wherein the tumor associated antigen is CD20, CD22, CEA, CSAp, AFP or MUC-1, or wherein the second antibody or fragment thereof is hMN-14, Mu-9, Immu31 or PAM 4.

15. The use of claim 1, wherein the anti-EGP-1 antibody or fragment thereof is covalently linked to a second antibody or fragment thereof.

16. The use of claim 1, wherein said anti-EGP-1 antibody or fragment thereof is a chimeric, humanized or human RS7 antibody or fragment thereof.

17. The use of claim 16, wherein the chimeric, humanized or human RS7 antibody or fragment thereof comprises the RS7 antibody light chain variable region CDR1(KASQDVSIAVA, SEQ IDNO: 28); CDR2(SASYRYT, SEQ ID NO: 29); and CDR3(QQHYITPLT, SEQ ID NO: 30) and RS7 heavy chain variable region CDR1(NYGMN, SEQ ID NO: 31); complementarity Determining Regions (CDRs) of CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO: 32) and CDR3(GGFGSSYWYFDV, SEQ ID NO: 33).

18. The use of claim 1, wherein said anti-EGP-1 antibody or fragment thereof comprises a human IgG1 constant region sequence.

19. The use of claim 1, wherein the disease is cancer.

20. The use of claim 1, wherein the cancer is a tumor of the lung, breast, bladder, ovary, uterus, stomach, or prostate.

21. The use of claim 1, wherein the lung tumor is a squamous cell carcinoma or adenocarcinoma.

22. Use of a chimeric, humanized or human anti-EGP-1 antibody or fragment thereof conjugated to a therapeutic agent in the manufacture of a medicament for the treatment of a disease in which diseased cells express EGP-1 antigen, wherein the anti-EGP-1 antibody or fragment thereof comprises a human IgG1 constant region.

23. The use of claim 22, wherein the therapeutic agent is selected from the group consisting of isotopes, drugs, toxins, immunomodulators, hormones, enzymes, rnases, antibodies, antibody fragments, growth factors, radionuclides, and antisense oligonucleotides.

24. The use of claim 23, wherein the radionuclide is selected from¹³¹I、¹²³I、¹²⁴I、⁸⁶Y、⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、^99mTc、^94mTc、⁹⁰Y、¹¹¹In、¹²⁵I、¹⁸⁶Re、¹⁸⁸Re、¹⁸⁹Re、¹⁷⁷Lu、⁶⁷Cu、²¹²Bi、²¹³Bi、²¹¹At、¹⁹⁸Au、²²⁴Ac、¹²⁶I、¹³³I、⁷⁷Br、^113mIn、⁹⁵Ru、⁹⁷Ru、¹⁰³Ru、¹⁰⁵Ru、¹⁰⁷Hg、²⁰³Hg、^121mTe、^125mTe、¹⁶⁵Tm、¹⁶⁷Tm、¹⁶⁸Ag、¹¹¹Ag、¹⁹⁷Pt、¹⁰⁹Pd、³²P、³³P、⁴⁷Sc、¹⁵³Sm、¹⁰⁵Rh、¹⁴²Pr、¹⁴³Pr、¹⁶¹Tb、¹⁶⁶Ho、¹⁹⁹Au、⁵⁷Co、⁵⁸Co、⁵¹Cr、⁵⁹Fe、¹⁸F、⁷⁵Se、²⁰¹Tl、²²⁵Ac、⁷⁶Br、⁸⁶Y、¹⁶⁹Yb、¹⁶⁶Dy、²¹²Pb and²²³Ra。

25. the use of claim 23, wherein said Rnase is onconase.