Title:  Recombinant alpha-fetoprotein hybrid cytotoxins for treating and diagnosing cancers

United States Patent:  6,534,479

Issued:  March 18, 2003

Inventors:  Murgita; Robert A. (Montreal, CA)

Assignee:  Martinex R & D Inc. (Montreal, CA)

Appl. No.:  758757

Filed:  December 3, 1996

Disclosed is a method of inhibiting a neoplasm in a mammal, the method includes administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein.

SUMMARY OF THE INVENTION

In general, the invention features compositions and methods for the protection, treatment, and diagnosis of neoplasia, in particular, cancer. The invention is based on my discovery that unglycosylated recombinant human alpha-fetoprotein made in a prokaryote (e.g., E. coli) is useful for treating and diagnosing mammals with neoplasms, especially malignant tumors, such as breast or prostate carcinomas, and other carcinomas caused by a proliferation of malignant cells which express receptors which are recognized by recombinant human alpha-fetoprotein.

In one aspect, the invention features a method of inhibiting a neoplasm in a mammal (e.g., a human patient), involving administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or an anti-neoplasm fragment or analog thereof. Preferably, the neoplasm is a malignant tumor (e.g., a breast tumor or a prostate tumor); and the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated. In preferred embodiments, the cells of the neoplasm express a receptor which is recognized by the recombinant human alpha-fetoprotein. Such a neoplasm is generally a carcinoma such as an adenocarcinoma or a sarcoma. In preferred embodiments, the neoplasm proliferates in response to a hormone, e.g, estrogen or androgen. Preferably, administration of recombinant human alpha-fetoprotein inhibits the proliferation of cells of the neoplasm or, alternatively, kills cells of the neoplasm in the mammal. The method further includes administering to the mammal a chemotherapeutic agent.

In another aspect, the invention features a method of protecting a mammal from developing a neoplasm, involving administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein. Preferably, the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated.

In another aspect, the invention features a hybrid cytotoxin including a recombinant human alpha-fetoprotein (or a fragment or analog thereof) linked to a cytotoxic agent. Examples of such cytotoxic agents include, without limitation, diphtheria toxin, Pseudomonas exotoxin A; ricin and other plant toxins such as abrin, modeccin, volkensin, viscumin; chlorea toxin (produced by Vibrio cholerae bacteria); the so-called "Shiga-like" toxins (produced by E. coli and other enteric bacteria); Salmonella heat-labile enterotoxin; and E. coli heat-labile enterotoxin. In other preferred embodiments, the cytotoxic agent is non-proteinaceous. Examples of such non-proteinaceous cytotoxic agents include, without limitation, anti-cancer agents such as doxorubicin, as well as .alpha.-emitting radionuclides such as astatine and .beta.-emitting nuclides such as yttrium. Preferably, the cytotoxic agent of the hybrid cytotoxin is linked by a peptide bond to the recombinant human alpha-fetoprotein, and the hybrid toxin is produced by expression of a genetically engineered hybrid DNA molecule. In other preferred embodiments, the cytotoxic agent of the hybrid cytotoxin is a protein; such a cytotoxic agent is chemically conjugated to the recombinant human alpha-fetoprotein.

In other aspects, the invention features a detectably-labelled recombinant human alpha-fetoprotein or a detectably-labelled fragment or analog thereof capable of binding to a human neoplastic cell. Preferably such a molecule is labelled with a radionuclide, e.g., technetium-99 m, iodine-125, iodine-131, or indium. Other detectable labels include, without limitation, enzymes, fluorophores, or other moieties or compounds which emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate or, alternatively, the detectable signal can be an epitope recognized by an antibody (e.g., an epitope of alpha-fetoprotein or an epitope which is specifically engineered into the recombinant alpha-fetoprotein such as the HA or myc epitopes). Preferably, the molecule targets a malignant tumor (e.g. a breast tumor, a prostate tumor, or a carcinoma) which express a receptor which is recognized by the recombinant alpha-fetoprotein (or fragment or analog thereof). Typically, such recombinant alpha-fetoprotein is produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated.

Detectably-labelled recombinant human alpha-fetoprotein is useful for methods of imaging a neoplastic cell-containing region in a human patient in vivo. In general, the method involves: (a) providing a detectably-labelled molecule of recombinant human alpha-fetoprotein (or a fragment or analog thereof); (b) administering the molecule to the patient; (c) allowing the labelled molecule to bind and allowing unbound molecule to be cleared from the region; and (d) obtaining an image of the neoplastic cell-containing region. Preferably, the region is the breast or is the prostate. In other preferred embodiments, the region, without limitation, is liver tissue, is lung tissue, is spleen tissue, is pancreatic tissue, is brain tissue, is lymph tissue, or is bone marrow. Preferably, the image is obtained using dynamic gamma scintigraphy.

Detectably-labelled recombinant human alpha-fetoprotein (or fragment or analog thereof) can also be used in a method for diagnosing a neoplasm in a mammal (e.g., a human patient). Such a method includes: (a) contacting the biological sample with the detectably-labelled molecule of recombinant human alpha-fetoprotein; and (b) detecting the label bound to the sample, where the detection of label above background levels is indicative that the patient has a neoplasm. Preferably, the method involves a biological sample including cells fixed and sectioned prior to the contacting step, and the label bound to the sample is bound to areas corresponding to the cell membrane of the cells. In preferred embodiments, the biological sample is from the breast or prostate of a human patient.

Detectably-labelled recombinant human alpha-fetoprotein (or fragment or analog thereof) can also be used in a method for detecting a neoplasm a mammal in vivo. Such a method includes: (a) administering a diagnostically effective amount of the detectably-labelled molecule of recombinant human alpha-fetoprotein; and (c) detecting the presence of the detectable label bound to a tissue of the mammal, where an amount of label above background levels is indicative of the presence of the neoplasm in the mammal. In preferred embodiments, the method involves a human patient suspected of having a breast cancer, and the tissue is breast tissue. In other preferred embodiments, the method involves a human patient suspected of having a prostate cancer, and the tissue is prostate tissue. Preferably, the detectably labelled recombinant human alpha-fetoprotein is linked to a radionuclide (e.g., technetium-90) and the detection step is accomplished by radioimaging (e.g., dynamic gamma scintigraphy).

In another aspect, the invention features kits for detecting a neoplasm or any cell expressing a receptor which is recognized by recombinant human alpha-fetoprotein (or a fragment or analog thereof) in vivo, in situ or in vitro. In general, the kits include a recombinant human alpha-fetoprotein which is recognized by a neoplasm, and which may be detectably labeled. If the recombinant human alpha-fetoprotein is unlabelled, a second reagent containing a detectable label (e.g. a radionuclide such as technetium-90, iodine-125, iodine-131, or indium) is preferably included. Where the detectable label is an enzyme, the kit further includes a substrate reagent for the enzyme. The kit may also include a reagent for linking the detectable label to the recombinant alpha-fetoprotein. In another embodiment, the kit for detecting a neoplasm or any unwanted cell expressing a receptor which is recognized by recombinant human alpha-fetoprotein (or a fragment or analog thereof) includes a reagent containing an antibody which specifically binds the recombinant human alpha-fetoprotein and a reagent including a detectably labeled recombinant human alpha-fetoprotein that is specifically bound by the anti-alpha-fetoprotein antibody. Preferably, the recombinant human alpha-fetoprotein of the kit is produced in a prokaryotic cell (E. coli) and is unglycosylated.

By "neoplasm" is meant any unwanted growth of cells serving no physiological function. In general, a cell of a neoplasm has been released from its normal cell division control, i.e., a cell whose growth is not regulated by the ordinary biochemical and physical influences in the cellular environment. In most cases, a neoplastic cell proliferates to form a clone of cells which are either benign or malignant. Examples of neoplasms include, without limitation, transformed and immortalized cells, tumors, and carcinomas such as breast cell carcinomas and prostate carcinomas.

By "therapeutically effective amount" is meant a dose of unglycosylated recombinant human alpha-fetoprotein or an anti-neoplasm fragment or analog thereof capable of inhibiting the proliferation of a neoplasm.

By "diagnostically effective amount" is meant a dose of detectably-labelled recombinant human alpha-fetoprotein or a detectably-labelled fragment or analog thereof that can be detected within a targeted region in a mammal (e.g., a human patient).

By "recombinant human alpha-fetoprotein" is meant a polypeptide having substantially the same amino acid sequence as the protein encoded by the human alpha-fetoprotein gene as described by Morinaga et al., Proc. Natl. Acad. Sci., USA 80: 4604 (1983). The method of producing recombinant human alpha-fetoprotein in a prokaryotic cell is described in U.S. Pat. No. 5,384,250.

The use of recombinant human alpha-fetoprotein for the treatment and diagnosis of cancer offers a number of advantages. For example, rHuAFP can be administered directly to a tumor site. Recombinant HuAFP can also be chemically defined and synthesized, and prepared in large quantities using the techniques of recombinant DNA. Moreover, unlike conventional cancer chemotherapies and radiotherapies, recombinant human alpha-fetoprotein causes minimal side effects such as nausea, vomiting, and neurotoxicity. Accordingly, relatively high doses of rHuAFP can be safely administered.

The diagnostic methods of the invention are advantageous since in that they allow for rapid and convenient diagnosis of a neoplasm. For example, the use of rHuAFP as a diagnostic agent (e.g., by radioimaging using scintigraphy) is especially advantageous for real time imaging of cancer in both pre-surgical or intraoperative localization and staging of a cancer, e.g., breast cancer, as well as during post-surgical examinations. Using such diagnostic procedures permits non-invasive determination of the presence, location, or absence of a neoplasm which is advantageous for monitoring the condition of a patient.

Claim 1 of 4 Claims

What is claimed is:

1. A hybrid cytotoxin consisting essentially of a recombinant human alpha-fetoprotein (SEQ ID NO:2) or a fragment thereof selected from the group comprsing at least one of Domain I (SEQ ID NO:3), Domain II (SEQ ID NO:4), Domain III (SEQ ID NO:5), Domain I+II (SEQ ID NO:6), Domain II+III (SEQ ID NO:7), and rHuAFP Fragment I (SEQ ID NO:8) linked to a cytotoxic agent.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.